Western Blot Analysis of Phosphorylated CagA

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)].