Roti pvdf membrane

Cells were trypsinized and spinned down; pellets were dissolved in Laemmli extraction buffer at 95°C. Protein extracts of 2×10⁵ cells per lane were subjected to a 10% Tris/HCl sodium dodecyl sulfate-polyacrylamide gel electrophoresis [19] (link) and transferred to a Roti® PVDF membrane (Roth). After blocking, the membrane was incubated with anti-V5 monoclonal antibody (Life Technologies, Darmstadt, Germany) diluted 1∶2,000 at 4°C for 16 h. Washings were performed with PBS supplemented with 0.3% Tween-20 at room temperature (RT). Peroxidase–conjugated goat anti-mouse IgG (Sigma-Aldrich, diluted 1∶500) was incubated for 1 h at RT followed by 5× washing. The membrane was incubated with ECL™ Prime Western Blotting Reagents (GE Healthcare, Freiburg, Germany) for 5 min in the dark, and chemiluminescence was analyzed after 5 sec exposure time with the LumiImager F1 system (Roche Diagnostics) and LumiAnalyst program. Protein sizes were compared to PageRuler Plus Prestained Protein Ladder (Thermo Scientific).

For Western blot analysis, the explants of approximately 100 mg were cut from the leaves using a cylindrical metal punch and ground in 500 μl of 2-fold SDS-PAGE loading dye. After 10 min heat denaturation at 95 °C, the samples were separated in a 12% polyacrylamide gel and transferred onto a Roti-PVDF membrane with pore size 0.45 µm (Roth, Germany). The hybridization was carried out with anti-GFP mouse monoclonal primary antibody and goat anti-mouse secondary antibody fused with horseradish peroxidase (both from Santa Cruz Biotechnology; 1:10,000 dilution). For detection, the CheLuminate-HRP PicoDetect kit (AppliChem, Germany) was applied.

Infected HEK293 and AGS cell lines were harvested using cell scrapers, and heated at 95 °C for 5 min in 1× Laemmli buffer. Separation of proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on gels with 6–10% polyacrylamide followed by Western blot analysis using ROTI®PVDF membranes (Carl Roth, Karlsruhe, Germany). The membranes were incubated for 1 h at 20 °C with TBS-T buffer (140 mM NaCl, 25 mM Tris–HCl pH 7.4 and 0.1% Tween-20) including either 3% BSA or 5% skim milk [45 (link)] to block non-specific binding sites. For detection, the following antibodies were used: mouse monoclonal antibody to GAPDH was obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Phosphorylated and total CagA proteins were identified by successive probing of the blots with the mouse monoclonal α-pan-phosphotyrosine antibody PY99 (Santa Cruz Biotechnology) and rabbit polyclonal antibody against CagA (Austral Biologicals, San Ramon, USA) [46 (link)]. Polyvalent horseradish peroxidase (HRP)-coupled secondary goat antibodies were used to detect mouse and rabbit primary antibodies (Thermo Fisher Scientific, Massachusetts, USA). Subsequently, the blots were visualized using the ECL Prime chemiluminescence kit from GE Healthcare as described [47 (link), 48 (link)].

Cells were washed with ice cold PBS and scraped off in lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing protease and phosphatase inhibitors (Complete Mini EDTAfree and PhosStop, both Roche Applied Science). The lysate was incubated on a rotating wheel at 4°C for 45 min and afterward cleared from nuclei and debris by centrifugation at 2000 × g and 4°C for 5 min.
Separation of proteins was performed by SDS-PAGE using a Mini PROTEAN system (Bio-Rad) or Novex NuPAGE SDS-PAGE Gel system (Life Technologies) and transferred onto Roti-PVDF membranes (0.45 μm, Roth) using a Mini TransBlot Electrophoretic Transfer Cell device (Bio-Rad). Precision Plus Protein Standard (Bio-Rad) was used as a marker. Membranes were blocked with 4% (w/v) milk (Roth) in TBST [50 mM Tris, 150 mM NaCl, pH 7.2, 0,1% (v/v) Tween 20] for 30 min at room temperature. Binding of primary antibodies was carried out either at 4°C over night or for 1 h at room temperature. Suitable secondary antibodies (coupled to horseradish peroxidase, Dianova) were incubated for 30 min at room temperature. All antibodies were diluted in blocking medium. Image acquisition was performed in a ChemiDoc XRS system using Imagelab software (Biorad).