CagA translocation assay

AGS cells were seeded in six-well plates as described above and incubated until ∼80% confluence. Helicobacter pylori strains to be assayed for CagA translocation into AGS cells were added at an MOI of 100 and infection allowed to proceed for 14 h. Non-adherent H. pylori cells were removed by aspiration and AGS monolayers were washed three times with PBS and then lysed using RIPA Buffer (25 mM Tris-HCl [7.5], 150 mM NaCl, 5mM EDTA, 1% Triton X-100, 0.1% SDS) with 2 mM Na₃VO₄ and Protease Inhibitor Cocktail (Thermo). Twenty micrograms of soluble protein was resolved on 7% TGX polyacrylamide gels (Bio-Rad) and western blotted to nitrocellulose. Translocated and tyrosine phosphorylated CagA was visualized using a rabbit monoclonal antibody to phospho-Tyrosine (Abcam), while total CagA was detected in identical blots, run simultaneously with identical protein loads, using a mouse monoclonal anti-CagA (Sigma-Aldrich). Detection utilized horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse IgG (Abnova) and SuperSignal West Pico Chemiluminescent Substrate (Thermo). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as a protein loading control using a mouse monoclonal anti-GAPDH (Sigma Aldrich).