[page 14↓]

2.  Materials and Methods

2.1. Materials

2.1.1. Chemicals

All chemicals used in this work were purchased from Calbiochem, CA, USA; J.T. Baker, Deventer, Holland; Merck, Darmstadt, Germany; R&D Systems Inc., MN, USA; Serva Electrophoresis GmbH, Heidelberg, Germany; Sigma-Aldrich Chemie GmbH, Munich, Germany; Sigma, MS, USA.

MiliQ 18 Ω water was used in all procedures if required.

2.1.2. Kits

QIAprep Spin Miniprep

Qiagen GmbH, Hilden, Germany

Annexin-V-FLUOS Staining Kit

Roche Diagnostics GmbH, Mannheim, Germany

Anti-HA Sepharose conjugate

Sigma, MS, USA

Apoptosis Sampler Kit

Cell Signaling Technology, Inc., MA, USA

Bcl-2 Family Antibody Sampler Kit

Cell Signaling Technology, Inc., MA, USA

Developer RP X-OMAT EX

Eastman Kodak Company, NY, USA

ECL Western Blotting Detection Kit

Amersham Biosciences GmbH, Freiburg, Germany

Fast-LinkTM DNA Ligation and Screening Kit

Biozym Diagnostik GmbH, Oldendorf, Germany

Fixer RP X-OMAT LO

Eastman Kodak Company, NY, USA

Immunoprecipitation Kit (Protein G)

Roche Diagnostics GmbH, Mannheim, Germany

PhosphoPlus Stat1 Antibody Kit

Cell Signaling Technology, Inc., MA, USA

Protease inhibitors Cocktail

Roche Diagnostics GmbH, Mannheim, Germany

QIAGEN Plasmid Midi and Maxi

Qiagen GmbH, Hilden, Germany

QuikChange Mutagenesis Kit

Stratagene, CA, USA

RediPack GST Purification Module

Pharmacia Biotech Inc., CA, USA

SequaGel XR

National diagnostics, GE, USA

SequiTherm EXCELTM II DNA Sequencing Kit

Biozym Diagnostik GmbH, Oldendorf, Germany

Ser/Thr Phosphatase Assay Kit 1

Upstate Biotechnology, NY, USA

Western Blot Recycling Kit

Alpha Diagnostic International, TX, USA


[page 15↓]

2.1.3.  Enzymes

Lyticase

Sigma-Aldrich Chemie Gmbh, Munich, Germany

Ampli Taq DNA Polymerase

Perkin Elmer, MA, USA

Ampli Taq Gold DNA Polymerase

Perkin Elmer, MA, USA

Restriction Endonucleases: EcoRI, BamHI, HindIII, XbaI,

Promega, Mannheim, Germany

2.1.4.  Antibodies

Anti-BRCA1 (H-1000)

Santa Cruz Biotechnology, Inc., CA, USA

Anti-cleaved caspase-3

Cell Signaling Technology, Inc., MA, USA

Anti-cleaved caspase-9

Cell Signaling Technology, Inc., MA, USA

Anti-GST antibody

Cell Signaling Technology, Inc., MA, USA

Anti-HA

Sigma, MS, USA

Anti-HA Sepharose Conjugate

Sigma, MS, USA

Anti-Histone 3

Cell Signaling Technology, Inc., MA, USA

Anti-H-rev107, rat (08B6)

C. Sers, Charité, Berlin, Germany; (Sers et al., 1997)

Anti-H-REV107-1 (315)

C. Sers, Charité, Berlin, Germany; (Sers et al., 2002)

Anti-IRF1

Santa Cruz Biotechnology, CA, USA

Anti-IRF2

Santa Cruz Biotechnology, CA, USA

Anti-p14.5,

G. Schmitz, University of Regensburg, Germany (Schmiedeknecht et al., 1996)

Anti-p21

Santa Cruz Biotechnology, CA, USA

Anti-pan-actin

Chemicon, CA, USA

Anti-PC4

R. Heilbronn, Free University Berlin, Germany (Weger et al., 1999)

Anti-PR65 (6F9)

Covance Research Products Inc., CA, USA

Anti-STAT1

Cell Signaling Technology, Inc., MA, USA

Anti-V5

Invitrogen, CA, USA

peroxidase-conjugate goat anti-mouse

Dianova, Hamburg, Germany

peroxidase-conjugate goat anti-rabbit

Cell Signaling Technology, Inc., MA, USA

peroxidase-conjugate goat anti-rabbit

Dianova, Hamburg, Germany


[page 16↓]

2.1.5.  Fluorophore-Labelled Antibodies

AlexaFluor 488 fragment of goat anti-mouse IgG (H+L)

MoBiTec, Göttingen, Germany

AlexaFluor 594 fragment of goat anti-mouse IgG (H+L)

MoBiTec, Göttingen, Germany

AlexaFluor 546 fragment of goat anti-rabbit IgG (H+L)

MoBiTec, Göttingen, Germany

AlexaFluor 594 fragment of goat anti-rabbit IgG (H+L)

MoBiTec, Göttingen, Germany

2.1.6. cDNA Library

Pre-made human kidney LexA cDNA library cloned into the pJG4-5 vector containing AD, and carrying the yeast TRP1 transformation marker for selection in Trp yeast (Clontech, San Diego, TX, USA).

2.1.7. Mammalian Cell Lines

A27/80

Human Ovarian carcinoma cell line, European Cell Culture Collection (ECACC), UK

COS-7

African green monkey kidney fibroblasts, American Type Culture Collection (ATCC), USA

FE-8

HRAS-transformed derivative of the immortalised non-tumorigenic rat fibroblasts 208F (Griegel et al., 1986)

FE-8 H-rev107

FE-8 cells harbouring H-rev107 cDNA under tetracycline-inducible promoter (Sers et al., 1997)

FE-8 pUHD

FE-8 cells harbouring an empty vector containing tetracycline-inducible promoter (Sers et al., 1997)

OVCAR-3

Human Ovarian carcinoma cell line, American Type Culture Collection (ATCC), USA

2.1.8.  E. coli Strains

KC8

pyrF, leuB600, trpC, hisB463

BD Biosciences, Clontech, CA, USA

XL2-blue

recA1 endA1 gyrA96 thi-1 hsdR17 suoE44 relA1 lac [F’ proAB laclqZΔM15 Tn10(Tetr) Amy Camr]a

Stratagene, La Jolla, Canada

Sure 2

e14 (McrA ) Δ (mcrCB-hsdSMR-mrr)171 endA1 supE44 gyrA96 thi-1 hsdR17 relA1 lac recB recJ sbcC umuC::Tn5(Kan r ) uvrC [F’ proAB laclqZΔM15 Tn10(Tetr) Amy Camr]a

Stratagene, La Jolla, Canada

B21

E. coli B F dem omp T hsdS(r B -m B -) gal

Pharmacia Biotech Inc., CA, USA


[page 17↓]

2.1.9.  Yeast Strains (OriGene Technologies, Inc., MD, USA)

EGY48

MATα trp1 his3 ura3 leu2::6 LexAop-LEU2 (high sensitivity)

RFY206

MATa trp1 Δ ::hisG his3 Δ 200 ura3-52 lys2 Δ 201 leu2-3 (mating strain)

2.1.10. Plasmids and Expression Constructs

ΔC107-ΔN

The expression construct was generated by PCR-amplification of a 375-bp fragment of ΔCH-REV107-1HA, using the ΔN-fw and ΔN-rv primers, followed by insertion of the PCR-product into the BamHI sites of a pcDNA3.1 plasmid

ΔC107-HWAY

The expression construct was generated using the QuikChange Mutagenesis Kit from the ΔCH-REV107-1HA expression vector, with the hway-fw and hway-rv primers

ΔC107-NCE

The expression construct was generated using the QuikChange Mutagenesis Kit from the ΔCH-REV107-1HA expression vector, with the nce-fw and nce-rv primers

ΔCH-REV107-1HA

The ΔCH-REV107-1HA fragment was created by PCR-amplification of 454-bp of H-REV107-1 cDNA including 49 bp of 5’-untranslated region, and 405 bp of a coding region. The 107HA-fw, and 107HA-rv reverse primer containing sequence encoding HA-epitope were used. The amplified fragment was cloned into the BamHI and XbaI sites of a pcDNA3.1 plasmid

107-GST

The ΔCH-REV107-1 fragment was obtained from H-REV107-1 expression vector using PCR-amplification with 107-TH-fw and 107-TH-rv primers. PCR product was cloned into the BamHI sites of a pGE-2TK plasmid

BRCA1

Full length BRCA1 cDNA cloned into pcDNA3.1 expression vector was kindly provided by T. Ouchi, The Mount Sinai School of Medicine, NY, USA

EGFP (enhanced green fluorescent protein)

Clontech, San Diego, TX, USA

H-REV107-1 full length expression vector

pcDNA3.1 expression plasmid contains the complete open reading frame, 62 bp of 5’-untranslated region and 174 bp of 3’ untranslated region of the H-REV107-1 cDNA (Husmann et al., 1998)


[page 18↓]

H-REV107-1V5

The expression construct was generated by PCR-amplification of the full length H-REV107-1 cDNA using 107-HA-fw and 107-rv primers. The amplified fragment of 415 bp was ligated into the BamHI sites of a pEF6/V5 vector

pEF6/V5

Invitrogen, CA, USA

p8op-lacZ, URA3, AmpR

Clontech, San Diego, TX, USA

PC4-V5

The PC4 expression vector containing PC4 full length cDNA fused with V5 epitope, was purchased from GeneStorm Collection (Invitrogen, CA, USA)

PcDNA3.1

Invitrogen, CA, USA

ETF1-HA

The ETF1 full length cDNA was PCR-amplified from the yeast expression cDNA library, using pJG-Hindfw and BCO3-Hindrv primers. The fragment was ligated into the HindIII sites of a pcDNA3.1 expression plasmid

p14.5

The p14.5 full length cDNA was PCR-amplified from the yeast expression cDNA library, using pJG-Hindfw and BCO3-Hindrv primers. The fragment was ligated into the HindIII sites of a pcDNA3.1 expression plasmid

PcDNA3.1/GS

GeneStorm Collection (Invitrogen, CA, USA)

pEG202, HIS3, AmpR

OriGene Technologies Inc, MD, USA

pEG202-107

ΔCH-REV107-1 yeast expression plasmid containing 405 bp of the human H-REV107-1 cDNA with a deletion of 81 bp encoding a C-terminal membrane binding domain. The H-REV107-1 fragment was generated by PCR-amplification of H-REV107-1 cDNA using 107-TH-fw and 107-TH-rv primers, and cloned into the BamHI sites of a pEG202 plasmid

pGE-2TK

Pharmacia Biotech Inc., CA, USA

pJG4-5, TRP1, AmpR

OriGene Technologies Inc, MD, USA

PR65-V5

The PR65-expression vector containing full length PR65α-encoded cDNA fused with V5 epitope, was purchased from GeneStorm Collection (Invitrogen, CA, USA)


[page 19↓]

RARG-V5

The RARG-expression vector containing full length of RARG cDNA fused with V5 epitope was purchased from GeneStorm Collection (Invitrogen, CA, USA)

S100A6HA

The S100A6 full length cDNA was PCR-amplified from the yeast expression cDNA library, using pJG-Bamfw and BCO3-Xbarv primers. The fragment was ligated into the BamHI and XbaI sites of a pcDNA3.1 expression plasmid

STAT1

The STAT1-expression vector was kindly provided by S. Vinkemeier, Institute of Molecular Pharmacology, Berlin, Germany

2.1.11. Oligonucleotides (MWG-Biotech, Ebersberg, Germany)


[page 20↓]

Oligonucl.

Labelling

Sequence

ΔN-fw

 

5’ – CGG GAT CCC GAA GAT GGG AGA CC GAT TGA GAT TTT TCG – 3’

ΔN-rv

 

5’ – CGG GAT CCC GTT AGG CAT AAT CAG GGA CGT CAT AAG G – 3’

107HA-fw

 

5‘ – CGG GAT CCA TGC GTG CGC CCA TTC CAG AG – 3’

107-HA-rv

 

5’ – TTA GGC ATA ATC AGG GAC GTC ATA AGG ATA AGG ATA GAT GAT GAC ATC TCT GAC CTG G – 3‘

107-rv

 

5’ – CGG GAT CCT TAG ATG ATG ACA TCT CTG ACC TGG – 3’

BCO3

IRD800

5’ – GTC AAG TCT CCA ATC AAG GTT – 3’

BCO3-Hindrv

 

5’ – TCC GAA GTC AAG TCT CCA ATC AAG GTT – 3’

BCO3-Xbarv

 

5’ – AGA TCT GTC AAG TCT CCA ATC AAG GTT – 3’

BCO5

IRD800

5’ – TAA CGA TAC CAG CCT CTT GC – 3’

BCO5-5

IRD800

5’ – CGA GGA GTG CAA TGC – 3’

hway-fw

 

5’ – CGC CCT TTC TAC AGA GCC TGG GCC GCC TAT GTT GTT GGC GAT GG – 3’

hway-rv

 

5’ – CCA TCG CCA ACA TAG GCG GCC CAG GCT CTG TAG AAA GGG CG – 3’

nce-fw

 

5’ – CCA GTG AGA ACA GCG AGC ACT TTG TGA ATG AGC – 3’

nce-rv

 

5’ – GCT CAT TCA CAA AGT GCT CGC TGT TCT CAC TGG – 3`

pEF6-rv

 

5’ – CTA GAA GGC ACA GTC GAG GC – 3’

pGE-fw

IRD800

5’ – GGG CTG GCA AGC CAC GTT TGG TG – 3’

pGE-rv

IRD800

5’ – CCG GGA GCT GCA TGT GTC AGA GG – 3’

pJG-Bamfw

 

5’ – CGG GAT CCG AAG ATG GTC TAC CCT TAT GAT GTG CC – 3’

pJG-Hindfw

 

5’ – AAG CTT GCC ACC ATG GTC TAC CCT TAT GAT GTG CCA G – 3’

Sp6

IRD800

5’ – CGA TTT AGG TGA CAC TAT AG – 3’

T3

IRD800

5’ – AAT TAA CCC TCA CTA AAG GG – 3’

T7

IRD800

5’ – TAA TAC GAC TCA CTA TAG GG – 3’


[page 21↓]

2.2.  Methods

2.2.1.  Yeast Two-Hybrid System

2.2.1.1. Yeast Expression Vectors and General Procedure

To identify H-REV107-1 interacting proteins, a LexA-based Yeast Two-Hybrid system was used. We screened a human kidney cDNA library with a truncated form of the H-REV107-1 protein. For this purpose, a pEG202-107 expression vector was generated. We subcloned 405 bp of human H-REV107-1 cDNA, encoding the H-REV107-1 protein without 27 C-terminal aminoacids, into the pEG202 yeast expression vector in frame with the DNA-binding domain (DNA-BD). The pEG202 vector carries a yeast HIS3 marker for selection on HIS medium. The DNA-BD was provided by the prokaryotic LexA protein, which normally functions as a suppressor of SOS genes in E.coli when it binds to LexA operators (Ebina et al., 1993). A premade cDNA library, purchased from Clontech, contained inserts cloned into the pJG4-5 vector. This vector carries a yeast TRP1 transformation marker for selection on the TRP medium and a transcriptional activation domain under the control of a GAL1 promoter. The transcriptional activation domain (AD) was an 88-residue acidic E.coli peptide (B42) (Ma and Ptashne, 1987). To activate transcription from the GAL1 promoter, transformants must be grown in medium containing galactose (Gal) and raffinose (Raf) as the carbon source. Interaction between a target library-encoded protein fused with the AD, and H-REV107-1 fused with the DNA-BD resulted in the reconstitution of a novel transcriptional activator with binding affinity for LexA operators (Gyuris et al., 1993). Two reporter genes with up-stream LexA operators, the LEU2, integrated in the EGY48 genome, and the lacZ, located on the p8op-lacZ reporter plasmid, made an interaction phenotypically detectable. If the proteins did not interact with each other, the reporter genes were not transcribed (Fig. 4).

The EGY48 yeast host strain was first transformed with the p8op-lacZ reporter plasmid carrying the lacZ reporter gene and stored in SD/-Ura (Table 2) medium/25% glycerol for further experiments. These transformants were then used for the co-transformation with pEG202-107 and pJG4-5 library plasmids. After library transformation, cells were plated on a minimal synthetic dropout (SD) non-induction medium that selected for both pEG202-107 and the AD/library plasmid, but not for the interaction directly, to maximise plasmid copy number in each cell. After this step colonies were plated on the SD induction medium lacking leucine and containing X-gal for detecting protein-protein interactions (Fig. 5). Individual blue colonies were isolated by restreaking on the same medium and stored as master plates.


[page 22↓]

Fig. 4 Schematic diagram of the LexA Two-Hybrid System (BD Biosciences, Clontech, CA, USA)

The bait, the H-REV107-1 protein fused to the DNA-BD, and the pray, library proteins fused to the AD cannot activate transcription of the reporter genes by themselves. The Interaction between the bait and a pray leads to the close proximity of the binding and activation domains, and activation of transcription of the reporter genes.


[page 23↓]

Fig. 5 Screening of a AD fusion library for proteins that interact with H-REV107-1 (BD Biosciences, Clontech, CA, USA)


[page 24↓]

Table 2 Generated yeast strains and respective selective media

Plasmids used for the transformation of the yeast strain EGY48

Media used to select transformants

p8op-lacZ

SD/-Ura

pEG202-107

SD/-His

pJG4-5

SD/-Trp

pEG202-107, pJG4-5, p8op-lacZ

SD/-His/-Trp/-Ura

 

Media used to activate reporter genes

EGY48[pEG202-107, pJG4-5, p8op-lacZ]

SD/-His/-Leu/-Trp/-Ura to activate the LEU2 reporter gene

EGY48[pEG202-107, pJG4-5, p8op-lacZ]

SD/Gal/Raf/-His/-Trp/-Ura/X-gal to activate the LacZ reporter gene

EGY48[pEG202-107, pJG4-5, p8op-lacZ]

SD/Gal/Raf/-His/-Leu/-Trp/-Ura/X-gal to activate both the LEU2 and lacZ reporter genes

2.2.1.2. Yeast Strain Storage and Culturing

2.2.1.2.1. Storage

To prepare a yeast glycerol stock, a single colony was scraped from the agar plate. Then the cells were resuspended in 200–500 μl of YPD medium (or the appropriate SD medium) in a 1.5-ml microcentrifuge tube. After dispersing the cells by vortexing, sterile 50% glycerol was added to a final concentration of 25%. The vials were frozen at –70°C. Transformed yeast strains were stored in the appropriate SD dropout medium to keep selective pressure on the plasmid.

To recover frozen yeast, a small portion of the frozen glycerol stock was streaked onto a YPD (or appropriate SD) agar plate. The plate was incubated at 30°C until yeast colonies reach ~2 mm in diameter (this took 3–5 days). These colonies were used as a working stock. The plates were sealed with parafilm and stored at 4°C for up to two months.

2.2.1.2.2. Culturing

Fresh (<2-months old) colonies from the working stock plate were used. One large (2–3-mm diameter) colony or several small colonies were inoculated in 5 ml of medium, and vigorously vortexed for ~1 min to disperse the cells. The Suspension was incubated at 30°C for 16–18 hr with shaking at 230–270 rpm. This yielded a stationary phase culture (OD600 > 1.5). To obtain a mid-log phase culture, the overnight culture was transferred into fresh medium and incubated at 30°C for 3–5 hr with shaking (230–250 rpm) to produce an OD600 = 0.2–0.3.


[page 25↓]

2.2.1.3.  Yeast Transformation

2.2.1.3.1. Preparation of Fresh Competent Yeast

Several colonies, 2–3 mm in diameter were inoculated in 1 ml of YPD or SD, and vortexed vigorously for 5 min to disperse any clumps. Then the cells were transferred into a flask containing 50 ml of YPD or the appropriate SD medium and incubated at 30°C for 16–18 hr with shaking at 250 rpm to yield a stationary phase culture (OD600>1.5). 30 ml of this overnight culture were transferred to a flask containing 300 ml of YPD and incubated at 30°C for 3 hr with shaking (230 rpm) until the OD600 reached 0.4–0.6.

Cells were placed in 50-ml tubes and centrifuged at 1,000 x g for 5 min at room temperature (20–21°C). Cell pellets were resuspended in H2O, then pooled into one tube (final volume 25–50 ml) and Centrifuged again at 1,000 x g for 5 min at room temperature. The cell pellet was resuspended in 1.5 ml of freshly prepared, sterile 1X TE/1X LiAc.

2.2.1.3.2. Transformation

For small scale transformation 0.1 μg of p8op-lacZ plasmid DNA and 0.1 mg of salmon testes carrier DNA were added to a fresh 1.5-ml tube and mixed.

For simultaneous co-transformation of pEG202-107 and library plasmids a large scale transformation was performed. The following amounts of DNA were used: 50 μg of pEG202-107 expression vector, 25 μg of library plasmid, and 2 mg of Salmon testes carrier DNA.

0.1 ml of yeast (1 ml for a large scale transformation) competent cells was added to each tube containing plasmid and salmon testes DNA and mixed well by vortexing. Then 0.6 ml of sterile PEG/LiAc solution (6 ml for a large scale transformation) were added to each tube and vortexed at high speed for 10 sec to mix, and incubated at 30°C for 30 min with shaking at 200 rpm. After the addition of 70 μl of DMSO (700 μl for a large scale transformation) the culture was mixed by gentle inversion.

A heat shock was performed for 15 min in a 42°C water bath and afterwards cells were chilled on ice for 1–2 min. Cells were centrifuged for 5 sec at 14,000 x g at room temperature (5 min 1000 x g for a large scale transformation). The cell pellets were resuspended in 0.5 ml of sterile 1X TE buffer (5 ml for a large scale transformation). 100 μl of the suspension (volume plated in large scale transformation) were than plated on SD agar plates 10 x 10 cm (15 x 15 cm for large scale transformation), that selected for the desired transformants.

For a small-scale transformation with the p8op[lacZ] plasmid only, the transformation was spread on the SD/-Ura plates. For large scale transformation the yeast suspension was spread on the SD/-His/-Trp/-Ura plates. Additionally, 100 μl of a 1:1000, 1:100, and 1:10 dilution were plated on 10 x 10 cm SD agar plates.


[page 26↓]

These plates were used as controls for transformation efficiency. Plates were incubated up-side-down, at 30°C until colonies appeared (2–4 days). To calculate the co-transformation efficiency, the colonies (cfu) growing on the dilution plate were counted (optimal 30-300 colonies on the plate with the dilution 1:100).

cfu x total suspension vol. (μl) / Vol. plated (μl) x dilution factor x amount of used DNA (μg)* = cfu/ /μg DNA

Sample calculation:

100 colonies grew on the 1:100 dilution plate (dilution factor = 0.01);

plating volume: 100 μl ; resuspension volume = 0.5 ml; amount of limiting plasmid = 0.1 μg

100 cfu x 0.5 ml x 103 μl/ml / (100 μl x 0.01 x 0.1 μg) = 5 x 105cfu/μg

If a small scale transformation was performed, for example to create an EGY48[p8op-lacZ] yeast strain, the largest colonies grown after transformation were picked and restreaked on the same selection medium for master plates. Then they were sealed with parafilm and stored at 4°C for 3–4 weeks. Alternatively they were stored at -70°C in SD/-Ura/25% glycerol.

If large scale transformation was performed the transformants were harvested as follows:

Plates were placed at 4°C for 3-4 hours to harden. Then 1 ml TE buffer was added to the surface of each plate. Colonies were scraped into the liquid using a sterile Pasteur pipette. All liquids were combined into a single sterile 50-ml tube and vortexed to resuspend the cells.

A Glycerol stock of the amplified yeast library was created by adding an equal volume of sterile 65% glycerol/MgSO4 solution. This stock can be stored at 4°C for one week or at -70°C up to 1 year.

2.2.1.4. β-Galactosidase Assay

To screen transformants for expression of a lacZ reporter, β-galactosidase assay was performed. We used two different methods: an in vivo assay and a colony-lift filter assay.

2.2.1.4.1. In Vivo Plate Assay Using X-gal – Containing Medium

Colonies from the master plates were replica plated on selection medium containing X-gal and BU salts, and incubated at 30°C for 4 days. Plates were checked every 12 hr (up to 96 hr) for the development of blue colour.


[page 27↓]

2.2.1.4.2.  Colony-Lift Filter Assay

In this assay fresh colonies (i.e., grown at 30°C for 2–4 days), 1–3 mm in diameter were used. A sterile Whatman filter was placed onto the surface of the plate, and gently rubbed, to improve attachment of the colonies to the filter. Three holes were poked through the filter in an asymmetric way to mark the orientation of the filter on the agar plate. When the filter was evenly wetted, it was carefully lifted off the agar plate with forceps and transferred (colonies facing up) to a pool of liquid nitrogen. Using the forceps, the filter was completely submerged for 10 sec. After the filter was frozen completely (~10 sec), it was removed from the liquid nitrogen and left to thaw at room temperature.

For each plate of transformants to be assayed, a fresh sterile Whatman filter was pre-soaked by placing it in 2.5–5 ml of Z buffer/X-gal solution in a clean 15 x 15 cm plate. (Avoid trapping air bubbles under or between the filters).

Then the first filter was carefully placed, colony side up, onto the pre-soaked filter to allow the X-Gal solution to get into contact with the colonies. The appearance of blue colonies was checked periodically.

The ß-galactosidase-producing colonies were identified by aligning the filter to the agar plate using the orientation marks. Corresponding positive colonies were picked from the original plates to fresh medium and incubated for 1–2 days to re-grow the colony.

2.2.1.5. Secondary Test of Positives Colonies

Positive colonies were re-tested at least once on the SD/-His/-Trp/-Ura plates. After incubation at 30°C for 4-6 days, colonies were replica plated on the SD/Gal/Raf/-His/-Leu/-Trp/-Ura induction medium to verify that they maintained the correct phenotype. Restreaked and re-tested colonies were collected on SD/-His/-Trp/-Ura plates, incubated at 30°C for 4-6 days. After colonies have grown, the plates were sealed with Parafilm and stored at 4°C for up to 4 weeks. For long term storage, a glycerol stock was prepared.

Recipes for Solutions and Buffers

YPD (rich medium), pH 5.8

20 g/l

Tryptone

10 g/l

Yeast extract

20 g/l

Agar (for plates only)

20 g/l

Glucose


[page 28↓]

SD-ura-his-leu-trp (selective medium)

6.7 g/l

Yeast nitrogen base w/o amino acids

20 g/l

Agar (for plates only)

0.6 g/l

-his-ura-trp-leu dropout mix

20 g/l

Galactose

20 g/l

Raffinose

20 g/l

Agar (for plates only)

80 mg/l

X-Gal

100 ml/l

10 x BU salts

All media were sterilised by autoclaving 120°C 20 min. The galactose, raffinose, X-Gal, and 10 x BU salts solutions were filter sterilised and added after autoclaving.

10 x BU salts (100 ml), pH 7.0

7 g

Sodium phosphate (dibasic)

3 g

Sodium phosphate (monobasic)

To prepare other SD selective media, for example lacking one of the aminoacid, following stock solutions were used:

Trp

10 ml of 4 mg/ml stock per litter of medium (0,04 mg/ml final concentration)

Ura

5 ml of 4 mg/ml stock per litter of medium (0,02 mg/ml final concentration)

Leu

15 ml 4 mg/ml stock per litter of medium (0,06 mg/ml final concentration)

His

5 ml of 4 mg/ml stock per litter of medium (0,02 mg/ml final concentration)

Salmon testes carrier DNA

Salmon testes carrier DNA (sodium salt) was dissolved in water (10 mg/ml), and the solution was stirred on a magnetic stirrer for 2-4 hours at the room temperature. Then the solution was extracted with phenol and with phenol : chloroform (1:1). The aqua phase was transferred into a new tube and sheared by passing 12 times rapidly through a 17-gauge hypodermic needle. The DNA was precipitated by adding 2 volumes of ice-cold ethanol.

DNA was recovered by centrifugation, and re-dissolved at a concentration of 10 mg/ml in water, boiled and stored in small aliquots at –20°C. Just before use, the solution was heated for 5 minutes in a boiling water bath and quickly chilled on ice.


[page 29↓]

PEG/LiAc solution

 

Final Conc.

10 ml solution

PEG 4000

40%

8 ml 50% PEG

TE buffer

1X

1 ml 10X TE

LiAc

1X

1 ml of 10X LiAc

Z buffer (pH 7.0), sterilised by autoclaving

16.1 g/l

Sodium phosphate (dibasic)

5.50 g/l

Sodium phosphate (monobasic)

0.75 g/l

Potassium chloride

0.25 g/l

Magnesium sulphate

Stock solutions:

50% PEG3350 prepared with H2O

10 x TE: 0,1M Tris-HCl, 10 mM EDTA, pH 7.5, autoclaved

10 x LiAc: 1M lithium acetate (pH 7.5 , adjusted with acetic acid), autoclaved

65% glycerol/MgSO4 solution (sterilised by autoclaving)

 

Final Conc.

Glycerol

65% v/v

MgSO4

100 mM

Tris-HCl, pH 8.0

25 mM

X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) was dissolved in DMF (N,N- dimethyformamide) at a concentration of 20 mg/ml and stored in the dark at – 20°C.

2.2.1.6. Mating Test

To test positive clones isolated in a library screening for interaction with H-REV107-1 a, mating assay was performed. Two yeast strains of opposite mating type, EGY48 – MATa, and RFY206 – MAT α were used. Mating occurred when the haploid cells of opposite mating type came into a contact and fused into a diploid yeast strain.

The EGY48 cells were transformed with the p8opLacZ plasmid and with either the pJG4-5 vector without insert or with the pJG4-5 vector containing candidate inserts chosen after sequencing analysis. Transformants were selected on the SD/-Trp/-Ura plates. The RFY206 cells were transformed with pEG202 or pEG202-107. Transformants were selected on the SD/-His plates. Then the two strains were mixed on SD/-His/-Trp/-Ura/-Leu/X-gal plates, where they formed diploid cells in which H-REV107-1 and one of the potential interacting partners have the opportunity to interact and to activate the reporter genes.


[page 30↓]

For each candidate of the library plasmid the follow matings were done:

Plasmid in EGY48

Plasmid in RFY206

LacZ phenotype for a true positive

Leu2 phenotype for a true positive

pJG4-5 without insert

pEG202 without insert

White

no growth

pJG4-5 with library insert

pEG202 without insert

white

no growth

pJG4-5 with library insert

pEG202-107

Blue

growth of colonies

pJG4-5 with insert

pEG202-107

White

no growth

Mating procedure:

One colony (2-3 mm) of each type used for the mating analysis was picked and placed together in a 1.5-ml microcentrifuge tube containing 0.5 ml of YPD medium, vortexed and incubated at 30°C with shaking at 250 rpm overnight. An aliquot (20μl) of the mating culture was spread on 100-mm, SD/-His/-Trp/-Ura plates and incubated at 30°C for 3-5 days to allow diploid colonies to form visible colonies. Then the diploid transformants were replica plated on the SD/Gal/Raf/-His/-Leu/-Trp/-Ura induction plates to assay for LEU2 an LacZ expression. True positives were those library clones which exhibited activation of the reporter gene expression only in presence of pEG202-107 plasmid.

2.2.1.7. Yeast Plasmid Isolation

A large (2-4 mm) fresh (2-4-day old) yeast colony was inoculated into 0,5 ml of the appropriate SD liquid medium and vortexed vigorously to resuspend the cells. The culture was incubated at 30°C overnight with shaking at 230-250 rpm. Then cells were spun down by centrifugation at 14,000 x g for 5 min, the supernatant was poured off and pellets were resuspend in the residual liquid (total volume ~ 50 μl) by vortexing or pipetting up and down.

10 μl of lyticase (5 units/μl in TE buffer) was added to each tube, and cells were thoroughly resuspended by vortexing or pipetting up and down. Tubes were incubated at 37°C for 30-60 min with shaking at 200-250 rpm. Then 10 μl of 20% SDS was added to each tube, and vortexed for 1 min to mix. All samples were incubated for 15 min at –20°C, than thawed and vortexed again to ensure complete lysis of the cells. (If necessary, samples were stored frozen at –20°C). The volume of each sample was brought up to 200 μl in TE buffer (pH 7.0).


[page 31↓]

Then 200μl of phenol : chloroform : isoamyl alcohol (25:24:1) was added and vortexed for 5 min. After centrifugation at 14,000 x g for 10 min, the upper phase was transferred to a fresh tube, 8 μl of 10 M ammonium acetate and 500 μl of 95-100% Ethanol were added. The solution was mixed carefully and placed at –70°C for 1 hour. Afterwards the tubes were centrifuged at 14,000 x g for 10 min. The supernatant was discarded, the pellet was dried and resuspend in 20 μl of H2O. This solution was used for transformation into competent E.coli KC8.

2.2.1.8. Preparation of Electrocompetent E. coli KC8

To prepare electrocompetent E. coli KC8 cells 1l of a 2x YT medium was inoculated with 100 ml of a fresh overnight culture, and incubated at 37°C with shaking until the OD600 reached 0.5–0.7. Then the flask was chilled for 15-30 min on ice, and centrifuged at 3,000 x g for 10 min in a pre-chilled rotor. The cells were washed in half of the original volume of sterile ice cold 1 mM Hepes pH 7.0, then spun down by centrifugation at 3,000 x g for 10 min in a pre-chilled rotor, and washed a second time with 50 ml of sterile ice cold water. The cells were centrifuged, and the pellet was resuspended in 2 ml of sterile ice cold water. The prepared cells were used directly for electroporation.

2.2.1.9. Transformation of the Electrocompetent E. coli KC8 with Yeast Plasmids

To transform E. coli KC8 with yeast plasmids, 40 μl of electrocompetent cells were mixed with 3 μl of yeast plasmid DNA, and transferred into a pre-chilled micro-electroporation chamber. For electroshock the following conditions were used: 1700 Volts, 200 Ohms, pulse control units were set to 25 μF. Directly after the shock, 1 ml of freshly prepared SOC medium was added to every sample and incubated with shaking for 1 hour at 37°C. Then 200 μl bacterial culture from every transformation were plated on M9 Agar plates to select transformants containing a yeast library plasmid.

2 x YT medium

16 g/l

bacto-tryptone

10 g/l

bacto-yeast extract

5 g/l

NaCl

pH was adjusted to 7.0 and sterilised by autoclaving

SOC medium

20 g/l

bacto-trypton

5 g/l

bacto-yeast extract

0.58 g/l

NaCl

0.19 g/l

KCl

pH was adjusted to 7.0 with NaOH, and sterilised by autoclaving. Then 1 ml of 2 M Mg2+ stock and 1 ml of 2 M Glucose was added to 98 ml of autoclaved medium.


[page 32↓]

Stock solutions

2 M Mg2+ stock (for 100 ml solution):

20.33 g

MgCl2 6H2O

24.65 g

MgSO4 7H2O

H2O was added to 100 ml and autoclaved.

2 M Glucose : 36.04 g of glucose was diluted in 100 ml of H2O and filter sterilised.

M9 minimal medium

200 ml

5 x M9 salts

2 ml

1M MgSO4

20 ml

20% glucose

0.1 ml

1M CaCl2

20 g/l

agarose (for plates only)

The medium was sterilised by autoclaving for 15 min at 120°C, and cooled down to 50°C. Then the following components were added per 1 l of the M9 medium:

1 ml

ampicillin 50 mg/ml

1 ml

thiamine-HCl solution

100 ml

10 x /-Trp supplement dropout (SD)

5 x M9 salts stock solution

64 g/l

NaH2PO4 7H2O

15 g/l

KH2PO4

2.5 g/l

NaCl

5.0 g/l

NH4Cl

2.2.1.10. Yeast Protein Isolation

For each clone to be assayed 5 ml of overnight culture were prepared in the appropriate SD selection medium. As a negative control untransformed yeast was cultured in 5 ml of YPD medium. The overnight cultures were inoculated in 50 ml of YPD medium and incubated at 30°C with shaking at 250 rpm until OD600 reached 0.4-0.6 (4-8 hours).

Each culture was chilled by pouring it into a pre-chilled 100-ml tube halfway filled with ice, and centrifuged for 5 min at 1000 x g by 4°C. The supernatant was poured off, and pellets were resuspend in 50 ml of ice-cold water. Cells were again centrifuged for 5 min at 1000 x g by 4°C, and frozen immediately by placing them in liquid nitrogen or dry ice. The cells where either be stored at -70°C, or used directly for further preparation.


[page 33↓]

The cell pellets were thawed and resuspend in 100 μl of pre-warmed (60°C) cracking buffer. Because PMSF degrades rapidly, every 7 minutes 1 μl of 100 mM PMSF was added to each sample. The suspensions were transferred into 1,5-ml microcentrifuge tubes containing 80 μl of glass beads. Then the samples were heated for 10 min at 70°C, vortexed vigorously for 1 min, and centrifuged for 5 min at 14,000 x g by 4°C.

Supernatants were saved on ice (supernatant I), the pellets were incubated for 3-5 min at 100°C, vortexed, and centrifuged again. The supernatant II was combined with supernatant I of each sample. Proteins were boiled for 1 minute and immediately loaded on a SDS-PAGE. Alternatively proteins were stored at -70°C.

PMSF 100 mM stock solution

0.17 g of PMSF was dissolved in 10 ml isopropanol

Protease inhibitor solution (688 μl were prepared freshly before use)

Inhibitor

Stock solution

Final concentration

Pepstatin A

66 μl of 1 mg/ml stock solution

0.1 mg/ml

Leupeptin

2 μl of 10,5 mM stock solution

0.03 mM

Benzamidine

500 μl of 200 mM stock solution

145 mM

Aprotinin

120 μl of 2.1 mg/ml stock solution

0.37 mg/ml

Cracking buffer stock solution (100 ml)

48 g

Urea

8M

5 g

SDS

5% v/v

40 mg

Bromphenol blue

0.4 mg/ml

4 ml

1M Tris-HCl, pH 6.8

40 mM

20 μl

0.5 M EDTA

0.1 mM

Cracking buffer complete (prepared before use)

1 ml

Cracking buffer stock solution

10 μl

β-mercaptoethanol

70 μl

Protease inhibitor solution

50 μl

PMSF stock solution


[page 34↓]

2.2.2.  Bacterial Culture

2.2.2.1.  Routine Culturing and Storage Conditions

The E. coli cultures were routinely grown at 37°C on Luria-Bertani (LB) agar or in LB broth, containing Ampicillin (75 µg/ml), Kanamycin (30 µg/ml), or Zeocine (25 µg/ml).

For long-term storage, a fresh overnight culture was prepared: bacteria were streaked onto an agar plate of the respective selective medium, and incubated at 37°C overnight. Several colonies were transferred separately into a 10 ml? Falcon tube containing 2 ml liquid media, and incubated for 14-16 hours at 37°C with shaking at 180-200 rpm. Then 800 μl of bacterial culture were transferred into the storage tube and sterile glycerol was added to a final concentration of 20%. The vials were snap-frozen in liquid nitrogen, and stored at –70°C until use. To recover a bacteria culture, a small proportion of the frozen glycerol stock was streaked onto an LB agar plate, containing the appropriate antibiotic.

The plate was incubated overnight at 37°C. These colonies were used as a working stock. The plates were sealed with parafilm and stored at 4°C for up to four weeks. Alternatively a small portion of the frozen glycerol stock was transferred in 2 ml of LB broth containing the appropriate antibiotic, and incubated overnight at 37°C with shaking at 180-200 rpm.

LB Broth (Agar)

10 g/l

NaCl

10 g/l

Tryptone

5 g/l

yeast extract

20 g/l

agar (for plates only)

pH was adjusted to 7.0 with NaOH and H2O was added to a final volume of 1 litter.

To prepare agar plates, the LB agar was cooled to 50°C, appropriate antibiotics were added and petri dishes were poured (~15 ml/100-mm plate). If a β-galactosidase assay was done, LB agar plates were prepared with 80 μg/ml X-gal (5-bromo, 4-chloro-3-indol-β-D-galactopyranisode), and 20 mM IPTG (isopropyl-l-thio-β-D-galactopyranoside). IPTG was prepared in sterile water, and X-gal in dimethylformamide (DMF).

2.2.2.2.  Transformation

Competent E. coli XL2-blue or Sure2 were purchased from Stratagene. Transformation was performed according to the protocol supplied by the manufacturer. Competent cells were thawed on ice and mixed gently. For each transformation, 100 μl of the cells were transferred into a microcentrifuge tube and 2 μl of β-mercaptoethanol were added. After incubation on ice for 10 min, 10 ng of plasmid DNA were added, swirled gently and incubated on ice for 30 min.


[page 35↓]

NZY+ broth was pre-heated to a 42°C. Tubes with bacteria were heat-pulsed in a 42°C water bath for 30 seconds. Then the tubes were incubated on ice for 2 min, 900 μl of the pre-heated NZY+ broth were added to every tube and incubated at 37°C for 1 hour with shaking at 225-250 rpm. 100 μl of each transformation mixture was plated on the appropriate plates using a sterile spreader and incubated overnight at 37°C.

2.2.2.3. Mini-Preparation of Plasmid DNA

For mini-preparation of plasmid DNA from bacteria a QIAprep Spin Miniprep Kit from Qiagen was used. For isolation of ~10 μg of plasmid DNA, 2 ml overnight culture of E. coli in LB medium was prepared. Plasmid DNA was isolated according to the manufacturer’s recommendations. DNA was eluted in 30 µl of the EB buffer (10 mM Tris-HCl, pH 8.5) and stored at -20°C.

2.2.2.4. Large-Scale Preparation of Plasmid DNA

For a large-scale plasmid isolation a QIAGEN Plasmid Midi and Maxi Kit was used. Plasmid DNA was isolated according to the supplier’s manual, a 200 ml E.coli overnight culture was prepared for each experiment. An additional purification step was included in the protocol. The pellet, resuspended in 400 µl of EB buffer was precipitated with 0,7 volumes of 4 M NaAc and 2,5 volumes of ethanol, and incubated for 1 hour at –70°C.

Then the solution was centrifuged at 14,000 x g for 30 min at 4°C, the pellet was washed with 1 ml ice-cold 70% ethanol, and centrifuged a second time at 14,000 x g for 5 min at 4°C, dried and resuspend in 200 µl of EB buffer.

2.2.2.5. Measurement of DNA Concentration

Concentration of DNA was measured using a UV-Spectrophotometer (Hitachi) by the wave length of 260 nm. The Amount of DNA was calculated regarding that OD260 = 1 corresponds to 50 μg/ml double-stranded DNA.

2.2.3. Enzymatic Manipulation and Analysis of DNA

2.2.3.1. Digestion of DNA with Restriction Endonucleases

Digestion of DNA with restriction endonucleases was performed according to the recommendations of the manufacturer. For the digestion of 5-10 µg of DNA, 10U of enzyme were added with an appropriate buffer to the DNA in a final volume of 15 ul. Reactions were incubated for 2 hours if PCR-fragments were digested, or 4-6 hours if plasmid DNA was digested.


[page 36↓]

2.2.3.2.  DNA Ligation

For the ligation of cDNA fragments into plasmid vectors, a Fast-Link DNA Ligation and Screening Kit purchased from Biozym was used. Experiments were performed according to a protocol by the supplier. For ligations, 200 ng of a digested plasmid DNA were used. The amount of insert was calculated according to a molar ration 3:1 (insert : vector). The reactions were incubated for 1 hour at room temperature. To inactivate the Fast-Link Ligase the reaction was transferred to 70°C for 15 min. To determine the efficiency of ligation, 5 μl of the reaction mix were run on an agarose gel and visualised with ethidium bromide. If the ligation was successful, 1-5 μl of the reaction mix was used for transformation of E.coli.

2.2.3.3. Vector Dephosphorylation

Prior to ligation of a digested plasmid DNA with an insert, the protruding 5’-end of the vector was dephosphorylated using a HK Thermolabile Phosphatase (Biozym). The reaction was performed according to the recommendations of the supplier. For dephosphorylation of 10 μg plasmid DNA 10 units of the phosphatase were used.

Digestion proceeded for 2 hours at 30°C, then the phosphatase was deactivated at 65°C for 15 min.

2.2.3.4.  Polymerase Chain Reaction (PCR)

The PCR reactions were performed in a PCR Thermal Cycler (Techne). Amplification of DNA was performed according to the following protocol:

Template DNA

20,0 ng

10 x PCR buffer (including 15 mM MgCl2)

2.50 μl

dNTP (each 10 nM)

0.25 μl

5’- and 3’ – oligonucleotide primers

1.00 μl (each)

H20 filled up to

24.0 μl

AmpliTaq Gold (1U)

0.25 μl

The Following program was used to amplify inserts:

Activation of the AmpliTaq polymerase

95°C

12 min

 

Denaturation of double stranded DNA

95°C

30 sec

 

Annealing of oligonucleotides to the template DNA

Tan

30 sec

30 cycles

Elongation

72°C

60 sec

 

Extension

72°C

5 min

 

Chilling

4°C

1 hour up to overnight.


[page 37↓]

Annealing temperatures were calculated using further formula: Tan = Tm - 4°C, where Tm is the melting temperature of the primers used for amplification . Tm of each oligonucleotide primer was calculated: Tm = 4 x (G + C) + 2 x (A + T), where G – guanine, C – cytosine, T – thymine, A – adenine.

2.2.3.5.  Purification of PCR-Amplified Fragments of DNA

For purification of PCR-amplified DNA fragments a QIAquick PCR Purification kit from Qiagen was used. The purification was performed according to the supplier’s protocol. Purified PCR fragments were controlled using agarose gel electrophoresis.

2.2.3.6. Site-Directed Mutagenesis

To generate the Δ C107-NCE and Δ C107-HWAY mutants of the H-REV107-1 protein, in vitro site-directed mutagenesis was performed using a QuikChange Site-Directed Mutagenesis Kit (Stratagene). The basic procedure utilised the Δ CH-REV107-1HA-expression plasmid and two oligonucleotide primers (sense and antisense) harbouring the desired mutations.

The primers were annealed to the denatured template DNA (Δ CH-REV107-1HA-expression plasmid ) and subsequently extended during temperature cycling using a PfuTurbo DNA polymerase. The incorporation of the primer sequence resulted in the generation of a single stranded, mutated plasmid containing a staggered nick. To digest the parental DNA template, the Dpn I endonuclease, specific for methylated and hemimethylated DNA, was used. Afterwards, the doublestranded plasmid containing the mutated base pairs was transformed into competent bacteria and amplified.

Two mutated H-REV107-1 proteins were generated. The first contained the His/Ala exchange at position 23 (the Δ C107-HWAY mutant), the second contained the Cys/Ser exchange at position 112 (the Δ C107-NCE mutant). For each mutagenesis a primer pair (sense and antisense) of 30-35 bp was used. For the His/Ala exchange, primer pair 1 contained mutated nucleotides G and C (wild type C and A, respectively). For the Cys/Ser exchange, primer pair 2 contained mutated nucleotide A (wild type T).

1sense (His/Ala)5’ – CCT TTC TAC AGA G /C C /AC TGG GCC ATC TAT GTT GGC – 3’

1antisense (His/Ala) 5’ – GCC AAC ATA GAT GGC CCA GG /T C /G TCT GTA GAA AGG –3’

2sense (Cys/Ser)5’ – CCA GTG AGA ACA /T GCG AGC ACT TTG TGA ATG AGC – 3’

2antisense (Cys/Ser)5’ – GCT CAT TCA CAA AGT GCT CGC T /AGT TCT CAC TGG – 3’


[page 38↓]

The reaction was set the following way

5 μl – 10 x reaction buffer

1 μl – template DNA (10 ng)

1 μl – oligonucleotide primer 1ps/2ps (125 μg)

1 μl – oligonucleotide primer 1ns/2ns (125 μg)

1 μl – dNTP mix

H20 filled to 50 μl; Then 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) was added.

Cycling parameters:

Activation of the PfuTurbo DNA polymerase

95°C

30 sec

 

Denaturation of double stranded DNA

95°C

30 sec

 

Annealing of oligonucleotides to the template DNA

55°C

60 sec

16 cycles

Elongation

68°C

12 min

 

Chilling

4°C

1 hour up to overnight

Amplification was checked by electrophoresis of 10 μl of the product on a 1% agarose gel. If a band was visible on the gel, a digestion with the Dpn I restriction enzyme was performed to digest the parental non-mutated supercoiled double stranded DNA. 1 μl of the enzyme was directly added to each amplification reaction and incubated for 1 hour at 37°C.

For transformation of the amplified, mutated cDNA, 1 μl of the reaction mix was used. Several colonies were selected for plasmid preparation and sequencing analysis to control that selected cloned contained the desired mutations.

2.2.3.7. Sequencing

Sequencing analysis was performed on a LI-COR automated DNA sequencer using fluorescent primers labelled with the tricarbocyanine dye IRD800 at their 5’-end.

Gel components were pre-mixed the following way

30 ml

Sequagel XR (Biozym)

7.5 ml

Sequagel buffer (Biozym)

400 μl

DMSO

300 μl

10% APS

and polymerised for 45 min.

Cycle sequencing reactions were done using a SequiTherm ExcelTM II DNA Sequencing Kit-LC (Biozym)according to a protocol of the supplier:


[page 39↓]

The reactions were pre-mixed in the following way

2 μl

2 pmol IRD800-labelled primer

100-250 fmol

DNA template

7.2 μl

3.5X SequiTherm EXCEL II Sequencing Buffer

1 μl

SequiTherm EXCEL II DNA Polymerase (5 U/μl)

H20 filled to 17 μl of a total reaction volume

The pre-mix was distributed in four tubes, 4 μl in each, 2 μl of the SequiTherm EXCEL II-LC Termination Mix A/C/G/T was added to each tube.

The following parameter were used for a cycle-sequencing:

30 sec

95oC

 

15 sec

Tan

30 cycles

1 min

70oC

 

Each reaction was stopped with 3 µl of a Stop/Loading Buffer. Samples were then analysed by electrophoresis or stored at -20oC. The annealing temperature of the primers was used equal to their melting temperature, and calculated as described in 2.2.3.5.

The reaction tubes were heated for 3-5 minutes at ≥ 70°C to denature the samples, and chilled on ice. Then 1,2 µl/well were loaded onto a sequencing gel, and run for 5-6 hours. Results were analysed using a Datalogger user program (LI-COR).

2.2.3.8. Electrophoretic Separation of DNA

Gel electrophoretic separation of DNA was performed in 1,0 – 1,5% agarose gels run at 30-50V with 1 x TBE as a running buffer. Gels contained 0,2 µg/ml ethidiumbromid, samples were run with 6 x Blue Loading Dye (Promega).

10 x TBE

108 g/l

Tris Base

55 g/l

Boric Acid

20 ml/l

0.5M EDTA

2.2.3.9. Elution of DNA Fragments from a Gel

To elute DNA fragments from a gel, a QIAquick Gel Extraction-Kit (QIAGEN) was used. The DNA fragments were cut from the gel on UV-light, and handled as recommended by the supplier. DNA was eluted in 30 μl of elution buffer.


[page 40↓]

2.2.4.  Culturing of Mammalian Cells

2.2.4.1.  Routine Culturing

All mammalian cell lines were maintained at 37°C and 95% humidity in the presence of 5% CO2. Human epithelial ovarian carcinoma cell lines A27/80 and OVCAR-3, African green monkey kidney fibroblasts COS-7, and FE-8 cells (HRAS-transformed derivative of 208F rat fibroblasts described by (Griegel et al., 1986), were cultivated in Dulbecco’s modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum and 2 mM glutamine. Tet-inducible FE-8 transformants (Sers et al., 1997) were cultured in DMEM medium supplemented with 10% tetracycline-free fetal calf serum and 2 mM glutamine either in the presence or in the absence of 2 μg/ml doxycycline.

For sub-culturing of the cells, the medium was removed, and the cells were rinsed with 1 x PBS buffer. Then the buffer was removed and 2 ml of trypsin-EDTA solution was added per 75 cm2 culture flask. The flask was incubated for a few minutes at 37°C until the cells detached. 8 ml of a fresh culture medium was added, aspirated and dispensed into new culture flasks. Split ratio: 1:10 for A27/80, COS-7, FE-8, and OVCAR-3 cells, and 1:2 for the Tet-inducible FE-8 cells. Fresh medium was added every 2 to 3 days.

Trypsin-EDTA 10 x stock solution (Biochrom AG) contained 0,5 % Trypsin and 0,2 % EDTA. For use in cell culture the solution was diluted 1:10 with PBS, filter-sterilised, and frozen at -20°C in 80 ml aliquots. Before use, an aliquot was thawed in a water bath at 37°C and used as described above.

PBS 10 x stock solution (pH 7.2)

2,3 g/l

NaH2PO4

11,5 g/l

Na2HPO4

87,5 g/l

NaCl

2.2.4.2. Freezing and Thawing Procedure

Cells were allowed to grow until they were 70% confluent, then medium was removed, and the cells were rinsed with 1 x PBS buffer, and 2 ml of trypsin-EDTA solution was added per 75 cm2 culture flask. The flask was incubated for a few minutes at 37°C until the cells detached, then they were transferred into a 15 ml Falcon tube and centrifuged at 800 x g for 3 min at 25°C. The cell pellet was resuspended in 900 μl of culture medium, and 100 μl of DMSO was added, mixed carefully and transferred into a cryo-tube and placed into a cryo-container with fresh isopropanol. The cells were kept at –70ºC for 24 hours to allow them to freeze slowly. After this time, they were stored in liquid nitrogen.


[page 41↓]

Thawing of cells was performed quickly. Frozen cells were thawed in a 37ºC water bath for 1-2 minutes and transferred immediately in a 15 ml Falcon tube. Ten ml of pre-warmed culture medium were added drop-wise and cells were resuspended. Then the tube was centrifuged at 800 x g for 3 min at 25°C to remove DMSO. The cell pellet was resuspended in 10 ml of fresh medium, transferred into a 75 cm2 culture flask, and cultured as described above.

2.2.4.3. Cell Treatments

OVCAR-3 cells were allowed to grow in their culture medium until they were 70% confluent. Stock solutions of 100 nM OA, and 500 μM LY294002 were prepared in DMSO. The cells were treated with 0,5 nM and 10 nM OA, and 50 μM LY294002, respectively for 48 hours in their culture medium. Cells were treated for 1 hour with 2 nM OA for the phosphatase assay. Control plates were treated with the vehicle, DMSO.

2.2.4.4.  Transfection of Mammalian Cells

COS-7 and OVCAR-3 cells were transfected using FuGENE 6 transfection reagent (Roche) according to a protocol of the supplier. For transfection in 6-well plates 5 x 104 cells were seeded. Twenty four hours later, cells were transfected with 0,5 μg plasmid DNA and 1.2 μl FuGENE reagent per well. For transfection in 10 mm dishes 3 x 105 cells were seeded and cultured for 24 hours, then transfected with 3 μg of total plasmid DNAs and 7 μl FuGENE reagent.

To control the transfection efficiency, cells were transfected with an EGFP plasmid (enhanced green fluorescent protein; Clontech) and viewed on UV light 48 hours after transfection. The number of green fluorescent cells was visually estimated.

2.2.4.5. Colonie Formation Assay

The colony formation assays, was performed by Jacqueline Hellwig (Institute of Pathology, Berlin, Germany) as described by Sers et al., (2002). Shortly, 2x105 cells per well of a 6 well plate were transfected with 1.5 μg plasmid DNA using FuGENE 6 transfection reagent (Roche). Forty-eight hours post transfection, G418 was added at 0.6 mg/ml to the culture medium. Selection was continued for 10 to 14 days until colonies were visible. Colonies were fixed in methanol, stained in methylen blue and counted.

2.2.5. Apoptosis Assays

We used two different assays to measure apoptosis. One assay was based on the altered nuclear morphology visible in apoptotic cells, the second assay measured the expression of Annexin V on the outer cellular membrane during apoptosis.

2.2.5.1. DAPI-Staining of Apoptotic Nuclei

For immunofluorescence cells were grown on the glass coverslips. After fixation with 3% paraformaldehyde in 1 x PBS for 20 min.


[page 42↓]

Cells were permeabilised using 0.2% Triton X-100 in 1 x PBS for 1.5 min. Coverslips were incubated with 100 ng/ml DAPI in 1 x PBS/1% BSA for 30 min, washed 3 times with 1 x PBS and mounted with 2.3% DABCO.

The cells were viewed using a confocal microscope TCS (Leica), and photographed. A number of cells exhibiting a nuclear morphology characteristic for apoptosis was calculated.

PBS 10 x stock solution (pH 7.2)

2.3 g/l

NaH2PO4

11.5 g/l

Na2HPO4

87.5 g/l

NaCl

DAPI (4',6'-diamidino-2-phenylindole hydrochloride) stock solution

100 μg/ml diluted in sterile H2O and stored at -20°C.

DABCO (1,4 – 1,4-Diazabicyclo (2,2,2)-octane) solution

2.3% w/v DABCO diluted in Glycerol/0,2 M Tris pH 8.0, ratio 9:1.

2.2.5.2. Flow Cytometric Analysis of Annexin V

OVCAR-3 cells were harvested 48 and 72 hours after incubation with 10 nM OA or the vehicle DMSO by trypsin-EDTA treatment and washed twice with ice-cold PBS. Apoptotic cells were detected using an Annexin-V-FLUOS Staining Kit (Roche) based on the dual staining technique with Annexin V, specifically staining apoptotic cells, and propidium iodide, staining all damaged cells. The experiment was performed according to the recommendations of the supplier. In a FACScan automate (Becton Dickinson), 10 000 cells were analysed in channels FL-1 for detection of the Propidium iodide stained cells, and FL-3 to detect Annexin V stained cells. The experiment was performed by Cornelia Giseler (Institute of Pathology, Charité, Berlin, Germany). The fraction of apoptotic cells was calculated using CellQuest software.

2.2.6. Analysis of Proteins

2.2.6.1. Protein Isolation from Mammalian Cells

Cells were grown in 10 cm dishes, and harvested when they reached confluency of 70%. The Medium was aspirated and cells were washed 3 x with ice-cold PBS. Then the cells were incubated with TNE lysis buffer for 30 min on ice. The lysed cells were scraped off the plates, transferred into microcentrifuge tubes and centrifuged for 15 min at 14,000 x g at 4°C. The Supernatants were removed, and pellets were discarded. An equal volume of 2 x SDS Sample buffer was added to each sample, the samples were boiled for 10 min, then chilled on ice and microcentrifuged for 5 min. Aliquots were stored at –20°C until use.


[page 43↓]

TNE lysis buffer

10 mM

Tris-HCl, pH 7.8

150 mM

NaCl

1%

NP-40

1 mM

EDTA

1/20 ml

protease inhibitor complete TM tabs

2 x SDS Sample buffer

50 mM

Tris-HCl, pH 6.8

100 mM

DTT

2% w/v

SDS

10% w/v

Glycerol

0.1% w/v

bromophenol blue

Prior to Western Blot analysis of caspases, an alternative protocol was used for the preparation of cellular extracts. The cells were allowed to grow for 48 hours then the medium was aspirated, transferred into pre-chilled Falcon tubes, and kept on ice. The cells were washed 3 x with ice-cold PBS and scraped into PBS. Then the tubes with medium and cells in PBS were centrifuged for 5 min at 1,000 x g. The supernatants were discarded, and CHAPS cell extract buffer (one volume of cell pellet) was added to the cell pellets.

The cells were resuspended in CHAPS buffer, frozen and thawed three times to lyse them, and centrifuged at 14,000 x g for 15 min at 4°C. The Supernatants were transferred into fresh tubes, an equal amount of SDS-Sample buffer was added, boiled for 10 min, and chilled on ice. The aliquots were stored at -20°C until use.

Chaps cell extract buffer

50 mM

Pipes/NaOH, pH 6.5

2 mM

EDTA

0.1%

Chaps

5 mM

DTT

20 μg/ml

Leupeptin

10 μg/ml

Pepstatin

10 μg/ml

Aprotinin

1 mM

PMSF


[page 44↓]

2.2.6.2.  Subcellular Fractionation

Cells were seeded in 10 cm dishes and allowed to grow in their culture medium until they were 70% confluent. The medium was aspirated, cells were washed 3 x with ice-cold PBS, and incubated with hypotonic-lysis buffer (1 ml per 10 cm plate/2 x 106 cells) for 5-10 min on ice. Samples were transferred into Dounce Homogenizers, pre-chilled on ice and homogenised by repeated strokes to disrupt the cells. To pellet the nuclei cells were centrifuged at 1000 x g for 10 min at 4°C. Supernatants were transferred into Beckman tubes (13 x 15 mm), the pellets containing nuclei were saved on ice.

To recover membrane-associated proteins, the supernatants were centrifuged in a TLA 100.3 rotor at 100 000 x g for 30 min at 4°C. The supernatants containing cytoplasmic proteins were removed and transferred into Corex tubes, pellets were dissolved in 50μl ice-cold PBS and saved on ice.

To collect cytoplasmic proteins, a methanol/chloroform precipitation was used (Wessel and Flugge, 1984). To each supernatant prepared in the previous step, 4 ml pre-cooled methanol was added, then 1 ml pre-cooled chloroform was added and mixed, then 3 ml pre-cooled sterile water was added and mixed. The solution was centrifuged at 9 000 x g for 15 min at 4°C. The upper phase was discarded, and 3 ml methanol were added to each sample, mixed and centrifuged again 9 000 x g for 15 min at 4°C. The pellets were dried and dissolved in SDS sample buffer.

SDS-sample buffer was added to all fractions, samples were boiled for 10 min, chilled on ice, and stored at -20°C until use.

Hypotonic lysis buffer

10 mM

Tris-HCl, pH 8.0

0.1 mM

DTT

1 tab/20 ml

protease inhibitor complete TM tabs

2.2.6.3. Determination of Protein Concentration

The protein concentration was determined using the BSA Protein Assay (PIERCE) according to the recommendations of the supplier.

2.2.6.4. One-Dimensional SDS Gel Electrophoresis (PAGE)

Protein samples were separated by polyacrylamide gel electrophoresis (PAGE). 50 μg of protein extract for detection of H-REV107-1 protein, and 30 μg for detection of caspases were loaded on a 12 % gel, run 30 min at 65V, and 2,5 hours at 98V in 1 x running buffer.


[page 45↓]

Separating gel: 10 ml of a 12% gel

3.35 ml

H2O

2.5 ml

1,5 M Tris-HCl, pH 8,6

4 ml

30% acrylamide/bisacrylamide

100 μl

10% SDS

50 μl

APS

5 μl

TEMED

Stacking gel: 10 ml of 4% gel

6.1 ml

H2O

2.5 ml

1,5 M Tris-HCl, pH 8,6

1.33 ml

30% acrylamide/bisacrylamide

100 μl

10% SDS

50 μl

APS

10 μl

TEMED

5 x running buffer

15.1 g/l

Tris-base

75 g/l

Glycine

5 g/l

SDS

2.2.6.5. Western Blot Analysis

After gel electrophoresis was finished, the stacking gel was discarded, and the separating gel was immersed in transfer buffer for 15 min. A PVDF-membrane was immersed in methanol for 5 sec, then in water for 2 min with shaking on a rotor platform, then the membrane was incubated 15 min in transfer buffer. On a Bio-Rad Transblot 3 sheets of Whatman paper pre-wetted in transfer buffer, the PVDF membrane, the gel, 3 sheets of pre-wetted Whatman were assembled.

Proteins were transferred to the PVDF membrane during 35 min at 16 V. After the transfer was finished, the membrane was washed briefly in TBST buffer, and blocked for 1 hour in 15 ml of blocking solution at room temperature with shaking on a rotor platform.

The gel was stained in Comassie blue solution to control for equal loading of protein amounts and efficient transfer. Afterwards the gel was photographed and discarded.

After the blocking procedure, the membrane was washed 3 x 10 min in 15 ml TBST, and incubated overnight with the primary antibody in blocking solution. The next day the membrane was washed 3 x 10 min in 15 ml TBST, and incubated for 1 hour with the corresponding secondary antibody.


[page 46↓]

The membrane was washed again 3 x 10 min in TBST, and the signal were developed using an ECL kit (Amersham), according to the supplier’s recommendations.

To strip a membrane, a Western Blot Recycling kit (Alpha Diagnostic) was used. Membranes were incubated in stripping solution for 45 min up to 1 hour, and rinsed with blocking solution supplied with the kit 2 x 5 min. After an additional washing in TBST for 10 min the membrane was ready for re-probing with other antibodies.

Primary antibodies, corresponding secondary antibodies, and their dilutions


[page 47↓]

Primary antibody

Dilution

Secondary antibody

Dilution

Anti-cleaved caspase-3 (Cell Signaling Technology, Inc., MA, USA)

1:1000

peroxidase-conjugate goat anti-rabbit (Cell Signaling Technology, Inc., MA, USA)

1:2000

Anti-cleaved caspase-9 (Cell Signaling Technology, Inc., MA, USA)

1:1000

peroxidase-conjugate goat anti-rabbit (Cell Signaling Technology, Inc., MA, USA)

1:2000

Anti-HA (Sigma Aldrich, Inc., MO, USA)

1:10 000

peroxidase-conjugate goat anti-mouse (Dianova, Hamburg, Germany)

1:10 000

Anti-Histone 3 (Cell Signaling Technology, Inc., MA, USA)

1:1000

peroxidase-conjugate goat anti-rabbit (Cell Signaling Technology, Inc., MA, USA)

1:2000

Anti-H-rev107, rat (C. Sers, Charité, Berlin, Germany; Sers et al., 1997)

1:500

peroxidase-conjugate goat anti-rabbit (Dianova, Hamburg, Germany)

1:50 000

Anti-H-REV107-1 (C. Sers, Charité, Berlin, Germany; Sers et al., 2002)

1:500

peroxidase-conjugate goat anti-rabbit (Dianova, Hamburg, Germany)

1:50 000

Anti-GST antibody (Cell Signaling Technology, Inc., MA, USA)

1:1000

peroxidase-conjugate goat anti-mouse (Dianova, Hamburg, Germany)

1:10 000

Anti-IRF1 (Santa Cruz Biotechnology, CA, USA)

1:1000

peroxidase-conjugate goat anti-rabbit (Dianova, Hamburg, Germany)

1:50 000

Anti-IRF2 (Santa Cruz Biotechnology, CA, USA)

1:1000

peroxidase-conjugate goat anti-rabbit (Dianova, Hamburg, Germany)

1:50 000

Anti-p14.5, obtained from C. Kerkhoff, University of Regensburg, Germany (Schmiedeknecht et al., 1996)

1:150

peroxidase-conjugate goat anti-rabbit (Dianova)

1:50 000

Anti-p21 (Santa Cruz Biotechnology, CA, USA)

1:1000

peroxidase-conjugate goat anti-rabbit (Dianova, Hamburg, Germany)

1:50 000

Anti-pan-actin (Chemicon, CA, USA)

1: 5000

peroxidase-conjugate goat anti-mouse (Dianova, Hamburg, Germany)

1:10 000

Anti-PC4, obtained from R. Heilbronn, Free University Berlin, Germany (Weger et al., 1999)

1: 500

peroxidase-conjugate goat anti-rabbit (Dianova, Hamburg, Germany)

1:50 000

Anti-PR65 (Covance Research Products, Inc., CA, USA)7

1:1000

peroxidase-conjugate goat anti-mouse (Dianova, Hamburg, Germany)

1:10 000

Anti-STAT1 (Cell Signaling Technology, Inc., MA, USA)

1:1000

peroxidase-conjugate goat anti-rabbit (Cell Signaling Technology, Inc., MA, USA)

1:2000

Anti-V5 (Invitrogen, CA, USA)

1 : 5000

peroxidase-conjugate goat anti-mouse (Dianova, Hamburg, Germany)

1:10 000

Transfer buffer (pH 8.5)

25 mM

Tris-Base

0.2 M

Glycine

20 %

Methanol

TBST (pH 7.4)

8.8 g/l

NaCl

0.2 g/l

KCl

3 g/l

Tris-Base

500 μl/l

Tween-20

Blocking buffer 5% non-fat dry milk in TBST

Commasie blue 0.1% Comassie blue R-250 dissolved in 40% Methanol, 10% acetic acid.


[page 48↓]

2.2.7.  Protein interaction analysis

2.2.7.1. Glutathione-S-Transferase Fusion System

2.2.7.1.1. Production of GST-Fusion Protein in Bacteria

The GST gene fusion system (Pharmacia Biotech, Inc., CA, USA) was used to express an H-REV107-1 – GST fusion protein in E.coli (107-GST). For this purpose the H-REV107-1 cDNA encoding a truncated H-REV107-1 protein lacking the membrane binding domain (the ΔCH-REV107-1) was ligated into the pGE-2TK bacterial expression vector in frame with the Glutathione S-transferase. The E. coli B21 bacterial strain recommended and purchased by Pharmacia Biotech was used for the expression of the fusion protein. To maintain the pGE-2TK plasmid and the 107-GST – expression vector the E.coli Sure 2 strain was used as recommended by the manufacturer.

Preparation of competent B21 cells

A single colony of a fresh overnight culture was inoculated in 50 ml LB and incubated at 37°C with shaking at 250 rpm. Cells were grown to an OD600 of 0,4 – 0,5, this took approximately 2.5 – 3 hours. Cells were then centrifuged at 2500 x g for 15 min at 4°C. The cell pellet was gently resuspended in 5 ml of ice-cold TSS buffer, placed on ice and used for transformations within the next 30 min.

TSS buffer (Recipe for 100 ml), pH 6.5, filter sterilised

1 g

Tryptone

0.5 g

yeast extract

0.5 g

NaCl

10 g

Polyethylene Glycol (MW 3350)

5 ml

DMSO

5 ml

1 M MgCl2

Transformation of competent B21 cells

One ml of competent cells was mixed with 10 ng of the pGE-2TK plasmid or 10 ng of the 107-GST expression vector, and incubated on ice for 45 min. All samples were transferred into a water-bath and incubated for 2 min at 42°C, then chilled briefly on ice. Afterwards, 100 μl of the transformation mix were transferred into a microcentrifuge tube with 900 μl LBG medium (LB-medium containing 20 mM Glucose) and incubated for 1 hour at 37°C with shaking at 250 rpm. 100 μl of the transformed cells were plated onto LBG plates containing 100 μg/ml Ampicillin and incubated overnight at 37°C.


[page 49↓]

Screening of pGE-recombinant transformants for expression of the fusion protein

Several colonies of E.coli transformants were picked into separate tubes containing 2 ml 2xYTA medium. Liquid cultures were grown to an OD600 of 0,6-0,8 (3-5 hours) with shaking at 30°C. Expression of the fusion proteins was induced by adding 2μl of 100 mM IPTG to each sample. Incubation was continued for an additional 2 hours. Liquid cultures were transferred into fresh tubes and centrifuged for 5 sec in a microcentrifuge. Pellets were resuspended in 300 μl of ice-cold PBS, 10 μl were analysed on an SDS-PAGE. To visualise the GST-protein (made in control cells carrying the parental pGE-2TK vector) and the fusion protein (made in cells carrying the 107-GST – expression vector), the gel was stained with Commassie Blue. Alternatively, a Western blot analysis with an anti-GST antibody was performed. The GST-protein has a size of 29 kDa, the 107-GST protein of 35 kDa. Several transformants, synthesising the recombinant protein of the predicted size were stored as a glycerol stock, as described in chapter 2.2.2.1.

2.2.7.1.2. GST Pull-Down Assay

The interaction between bacterially expressed 107-GST fusion protein and PR65-V5, over-expressed in COS-7 cells, was analysed under cell-free conditions using a GST pull-down assay.

Preparation of the recombinant 107-GST and GST proteins

To prepare recombinant 107-GST and GST, overnight cultures containing E.coli clones harbouring the pGE-2TK plasmid, and the 107-GST expression vector were grown. The overnight cultures were diluted 1:100, and 100 ml of every sample were incubated during 2-5 hours with shaking at 250 rpm at 30°C. Then, expression of the fusion proteins was induced by the addition of 100 mM IPTG to a final concentration of 0,2 mM. Incubation was continued for 3 hours. Cells were pelleted by centrifugation at 8000 x g for 10 min at 4°C and resuspended in 10 ml PBS. To lyse the cells, they were sonicated, and 20% Triton X-100 solution was added to a final concentration of 1%. Cell debris was pelleted at 12000 x g for 10 min at 4°C. Supernatants were transferred into fresh tubes, and 100 μl of 50% freshly prepared slurry of Glutathione Sepharose 4B were added to each supernatant.

The required amount of 50% Glutathione Sepharose 4B was prepared according to the recommendations of the supplier. 2 ml of a Sepharose were used for the purification of 250 μg protein.

For binding of recombinant proteins to Sepharose beads, samples were incubated at room temperature with gentle agitation for 30 min. Afterwards the suspensions were centrifuged at 500 x g for 5 min, and supernatants were removed. The Glutathione Sepharose beads with the bound proteins were washed 3 times with ice-cold PBS and added to a freshly prepared COS-7 cell protein extract.


[page 50↓]

GST-pull down assay

For this purpose COS-7 cells were transiently transfected with a PR65-V5 expression plasmid and incubated for 48 hours post-transfection. The cells were lysed in GST-lysis buffer, cell lysates were clarified by centrifugation, and saved on ice in 1 mg aliquots. To each aliquot were added purified recombinant 107-GST, or as a negative control GST proteins bound to Glutathione Sepharose beads, prepared as described above. The samples were incubated overnight at 4°C on a rocking platform. Then they were washed 3 times with GST-lysis buffer and resolved in SDS-PAGE, following Western blot analysis with an anti-GST, and an anti-V5 antibodies.

GST-lysis buffer

50 mM

Tris-HCl, pH 7.5

150 mM

NaCl

1 x (per 10 ml)

protease inhibitor complete tabs, EDTA free

1%

Nonidet P40

10%

Glycerol

2.2.8. Co-Immunoprecipitation

COS-7 cells were transiently transfected as described in chapter 2.2.4.4 and incubated for 42 hours post-transfection. The cells were lysed in the appropriate lysis buffer for 5 min, transferred to a pre-chilled Dounce homogeniser, and homogenised by repeated strokes on ice to disrupt the cells. Homogenised suspensions were centrifuged at 12,000 x g for 10 min at 4°C. Equal amounts of the supernatant from each sample were transferred into microcentrifuge tubes, optimal volume 1 ml. To each sample were added 5 μl of anti-V5, alternatively, 5 μl of anti-HA antibody, and gently rocked for 4 hours at 4°C. Then 50 μl of protein G-agarose (Roche, Mannheim, Germany) were added, and incubated overnight on a rocking platform at 4°C.

Protein complexes were collected by centrifugation at 12,000 x g for 20 seconds in a microcentrifuge, and washed 3 times with lysis buffer. Traces of lysis buffer after the final wash were removed, and 30 μl of SDS Sample buffer were added to each sample, heated for 5 min at 100°C, and frozen at -20°C. Precipitated protein complexes were analysed by SDS-polyacrylamide gel electrophoresis and immunoblotting.


[page 51↓]

Lysis buffer (for co-immunoprecipitation of H-REV107-1 with P14.5, ETF1, PC4, and PR65).

50 mM

Tris-HCl, pH 7.5

150 mM

NaCl

1 x (per 10 ml)

protease inhibitor complete tabs, EDTA free

1%

Nonidet P40

10%

Glycerol

For precipitation of S100A6/Calcyclin CaCl2 and ZnCl2 were added to a final concentration of 2 nM.

Lysis buffer for co-immunoprecipitation of H-REV107-1 with RARG (NET buffer)

20 mM

Tris-HCl, pH 8.0

200 mM

NaCl

1 mM

EDTA

0.1%

Nonidet P40

10%

Glycerol

1 x (per 20 ml)

protease inhibitor tabs complete

The following components were used to enhance binding of the RARG protein to H-REV107-1. They were added to the cell lysates and incubated for 30 min on ice prior to the addition of the antibody.

1 μg/ml

double stranded DR5 RARE or DR5M oligonucleotides

1 μM

ATRA

1 μM

TTNPB

2.2.9. Immunofluorescence Analysis and Confocal Microscopy

For immunofluorescence analysis cells were grown on glass coverslips. After fixation with 3% paraformaldehyde in 1 x PBS for 20 min, cells were permeabilised using 0,2% Triton X-100 in 1 x PBS for 1,5 min, washed 3 x 5 min with PBS and incubated with the primary antibody.

The H-REV107-1 antiserum was diluted 1:2000, and anti-V5, anti-PR65, anti-PR36 antibodies were diluted 1:300 in PBS/1% BSA. Coverslips were incubated with primary antibodies for 2 hours at room temperature, then washed 3 x 5 min in PBS and incubated with the secondary antibodies. A goat anti-rabbit antibody AlexaFluor 594 (Molecular Probes, Eugene, OR) at 1:500, and a goat anti-mouse antibody AlexaFluor 488 (Molecular Probes, Eugene, OR) at 1:200 were used. For nuclear staining, cell were incubated with diamidinophenylindole (DAPI) and analysed using confocal microscopy (Leica TCS).


[page 52↓]

Colocalisation of H-REV107-1 with PR65, and ΔNΔC107-1 with PR65 was analysed using a Leica confocal microscope TCS SL. Colocalisation was semi-quantitatively evaluated using the standard application LCS program (Version 2.585), and the LCS Multicolor-Software (Leica) by Kerstin Lehmann (MetaGen, Berlin, Germany).

2.2.10. Phosphatase Assay

Phosphatase activity was measured by using para-nitrophenyl phosphate (p-NPP) as a substrate with the Phosphatase Assay Kit (Upstate Biotechnology, NY, USA). To investigate a potential effect of the H-REV107-1 protein, OVCAR-3 cells were transfected with H-REV107-1 expression plasmid or the control pcDNA3.1 vector without insert, and then starved for 24 hours. OVCAR-3 cells were pre-treated with or without 2 nM OA for 1 hour at 37°C. Then the cells were lysed in lysis buffer used for co-immunoprecipitation of H-REV107-1 with PR65. Cell lysates were clarified by centrifugation at 12.000 x g for 10 min. The clarified supernatants were incubated with the anti-PR65 antibody (Covance Research Products. Inc., CA, USA) and protein G-agarose (Roche, Mannheim, Germany) for 2 hours at 4°C. The immunoprecipitates were washed 2 times with lysis buffer and 1 time with assay buffer, resuspended in assay buffer containing 2.5 mM NiCl2, and 900 μg/ml pNPP, and incubated for 30 min at 37°C. The amount of para-nitrophenol produced by dephosphorylation was determined by measuring the absorbance at 405 nm.

Assay buffer

50 mM

Tris-HCl, pH 7.0

0.1 mM

CaCl2

  


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