Western Blot Analysis of Key Bacterial and Host Proteins

AGS wt and AGSΔcttn cells intended for SDS-PAGE were harvested with hot (95 °C) SDS-buffer, followed by separation of the proteins in the lysates, according to size via 6–8% polyacrylamide gels. The proteins were then transferred onto a PVDF membrane for Western blotting and probed with antibodies [69 (link)]. Prior to probing, the PVDF membranes were blocked with either 3% BSA or 5% non-fat dry milk in TBST, depending on the primary antibody and manufacturers instructions [70 (link)]. Primary antibodies used were specific against cortactin (Merck-Millipore, Darmstadt, Germany #05-180), GAPDH (Santa Cruz, Heidelberg, Germany, #SC-47724), CagA (Austral Biologicals, San Ramon, CA, USA, #HPP-5003-9), CagE [63 (link)], VacA [34 (link)], FlaA [71 (link)], RPTP-α (Santa Cruz, #SC-28907), integrin-β1 (Santa Cruz, #SC-H96), full-length caspase-3 (Cell Signaling Technology, Frankfurt, Germany, #9668) and active caspase-3 (Cell Signaling Technology, #9664). Secondary antibodies detected either mouse (Invitrogen, Darmstadt, Germany, #31446) or rabbit (Invitrogen, #31460) primary antibodies and were coupled to horseradish peroxidase. Development of Western blots was performed as previously described [72 (link)].