Western Blot

Preparation of samples

The BCA™ Protein Assay Kit was used to measure protein concentration according to the manufacturer’s protocol. The protein samples were diluted in dH2O to reach the same

concentration for each sample and boiled in 5x SDS loading buffer (62.5mM Tris-base pH 10, 10% (w/v) SDS, 5% (v/v) β-mercaptoethanol, 50% (v/v) glycerol, bromphenol blue) for 10min at 99°C. After a short centrifugation, the samples were used for Western Blot analysis or stored at -20°C.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was performed to separate the proteins according to their molecular weight. Acrylamide gels (12.5%, 10% or 7.5%) were prepared as follows:

Component Separating gel Stacking gel

12.5% 10% 7.5% 4% H2O 2.59ml 3.29ml 3.99ml 1.53ml Acrylamide (30%) 3.5ml 2.8ml 2.1ml 333µl 1.5M Tris-HCl, pH 8.8 2.1ml 2.1ml 2.1ml - 0.5M Tris-HCl, pH 6.8 - - - 625µl SDS (10%) 83µl 83µl 83µl 25µl APS (10%) 42µl 42µl 42µl 12.5µl TEMED 2.8µl 2.8µl 2.8µl 2.5µl

The protein samples were loaded in equal amounts and the gel was run at constant 15mA in running buffer (192mM glycine, 25mM Tris-base, 0.1% (w/v) SDS) using the Mini-PROTEAN® Tetra Cell system.

Materials & Methods 46

Protein transfer and detection

Transfer of the separated proteins to a nitrocellulose membrane was achieved by the Mini trans-Blot® Electrophoretic Transfer Cell for 1h at 100V in transfer buffer (192mM glycine, 25mM Tris-base, 20% (v/v) methanol). The membrane was then blocked for 1h in 5% (w/v) BSA or milk in T-PBS (0.1% (v/v) Tween 20 in PBS) on a shaker at RT and incubated overnight in primary antibody solution at 4°C while gentle shaking. The next day, the membrane was washed in T-PBS 3x 10min before adding peroxidase-labelled secondary antibody solution (1:5000). After 1h incubation time, the membrane was washed again 3x 10min and immunodetection was performed using Enhanced Chemiluminescence Detection Reagent A and B 1:1 (A: 3µl H2O2 in 5ml 0.1M Tris-HCl, pH 8.5; B: 50µl luminol and 22µl p-coumaric acid

in 5ml 0.1M Tris-HCl, pH 8.5) using the Fusion Solo device.

Table 2.15: Antibody dilutions used for Western Blot analysis.

Antibody Company blocking dilution

Primary antibody

β-Actin Clone AC-15, A1978 Sigma Aldrich Chemie GmbH, Steinheim, Germany

5% milk/ 5% BSA

1:1000 in T-PBS DLC1, PA5-18290 Thermo Fisher Scientific, Inc.,

Surrey, UK

5% milk 1:1000 in 2.5% milk DLC1 C-terminal, ab180697 Abcam plc, Cambridge, UK 5% milk 1:500 in 2.5% milk p44/42 MAPK (ERK1/2),

#4695

Cell Signaling Technology, Inc., Danvers, USA

5% milk 1:1000 in 2.5% milk FLAG, ab8112 Abcam plc, Cambridge, UK 5% milk 1:1000 in 2.5% milk FLAG, F1804 Sigma Aldrich Chemie GmbH,

Steinheim, Germany

5% milk 1:1000 in 2.5% milk GFP, #11 814 460 001 Roche Diagnostics GmbH,

Mannheim, Germany

5% milk 1:1000 in 2.5% milk HIF1α, #3716 Cell Signaling Technology, Inc.,

Danvers, USA

5% milk 1:1000 in 2.5% milk HSP 90α/β (H-114), sc-7947 Santa Cruz Biotechnology,

Inc., Heidelberg, Germany

5% milk/ 5% BSA

1:1000 in T-PBS MYL2 (C-17), sc-34490 Santa Cruz Biotechnology,

Inc., Heidelberg, Germany

5% milk 1:1000 in 2.5% milk NFκB, #3033 Cell Signaling Technology, Inc.,

Danvers, USA

5% milk 1:1000 in 5% BSA Phospho-NFκB, #8242 Cell Signaling Technology, Inc.,

Danvers, USA

Materials & Methods 47

Antibody Company blocking dilution

Primary antibody

Phospho-Myosin Light Chain 2 (Ser19), #3671

Cell Signaling Technology, Inc., Danvers, USA

5% milk 1:1000 in 5% BSA RhoA (26C4), sc-418 Santa Cruz Biotechnology,

Inc., Heidelberg, Germany

5% milk 1:1000 in 2.5% milk ROCKII, #610624 BD Biosciences, Heidelberg,

Germany

5% milk 1:1000 in 2.5% milk ROCK2 (phospho T249),

ab83514

Abcam plc, Cambridge, UK 5% BSA 1:500 in 1% BSA Secondary antibody

Anti-rabbit IgG, HRP-linked antibody, #7074

Cell Signaling Technology, Inc., Danvers, USA

5% milk/ 5% BSA

1:5000 in BSA/ milk Donkey anti-goat IgG, HRP-

linked Antibody, sc-2020

Santa Cruz Biotechnology, Inc., Heidelberg, Germany

5% milk/ 5% BSA

1:5000 in BSA/ milk ECL™ Anti-mouse IgG, HRP-

linked Antibody, NA931V

GE Healthcare UK 5% milk/ 5% BSA

1:5000 in BSA/ milk

2.2.3.4 (Co)-Immunoprecipitation

Immunoprecipitation (IP) was done to enrich a specific protein out of solution using an antibody that specifically binds to that antigen. The antibody needs to be coupled to a solid substrate. Co-immunoprecipitation (CoIP) was performed to analyze protein-protein interactions. This method is based on the interaction of an antibody with a known protein, which is suggested to be part of a protein complex. Co-IP aims to precipitate the entire protein complex out of solution and later to identify unknown components of the complex by immunoblot (IB).

To perform CoIP, cells were grown to 90% confluency on a TC-dish 60 and transiently transfected as described in 2.2.1.5. For hypotonic lysis, 1ml lysis buffer (10mM Tris-HCl, pH 7.4, 2mM EDTA, 2mM MgCl2, 1mM sodium orthovanadate, 1mM DTT, Protease Inhibitor

Complete®) was added. The cells were scraped and transferred to a 2.0ml tube. For complete lysis, the cells were incubated on ice for 20min, homogenized by pipetting up and down 20 times until foaming and centrifuged for 10min at 13200rpm at 4°C. All following steps were performed on ice. The supernatant was mixed with 10µl Protein A/G PLUS- Agarose and incubated for 1h at 4°C under rotation. After centrifugation for 10min at 13200rpm and 4°C, 80µl of the supernatant were separated as input controls and stored at -

Materials & Methods 48

20°C, whereas 400µl of the supernatant were mixed with 4µg primary antibody (GFP #11 814 460 001 for CagA constructs, DLC1 PA5-18290 for DLC1.1 and FLAG, ab8112 for DLC1.4). Furthermore, 400µl of each sample were left without antibody as negative controls. The samples were incubated overnight at 4°C under rotation. The next day, 60µl Protein A/G PLUS-Agarose were added and incubated for 2h at 4°C under rotation. Beads were pelleted by centrifugation for 1min, 13200rpm at 4°C followed by three washing steps with 1ml wash buffer (lysis-buffer supplemented with 150mM NaCl) and subsequent centrifugation for 2min and 13200rpm at 4°C. For elution, the supernatant was mixed with 50µl of 100mM glycine (pH 2.2) and incubated on ice for 2min. The reaction was stopped by adding 10µl of 1.5mM Tris-HCl (pH 8.8). The eluate was collected after further centrifugation for 1min, 13200rpm at 4°C and supplemented with 10µl 5x SDS-loading buffer. The input controls were supplemented with 20µl 5x SDS-loading buffer and all samples were boiled for 10min at 99°C for Western Blot analysis.

To perform IP, frozen mouse liver tissue (2-3mm³) was added to 1ml hypotonic lysis buffer for tissue (1M Tris pH7.4, 0.5M EDTA, 1M MgCl2, 1mM sodium orthovanadate, 1mM DTT,

Protease Inhibitor Complete®). The tissue was homogenized using the Homogenisator IKA® T10 basic ULTRA-TURRAX® and incubated on ice for 1h. After resuspension and centrifugation for 10min at 4°C and 13200rpm, the supernatant was transferred into a new tube. All following steps were performed on ice according to the protocol for CoIP.