FIG. 3.

Immunoprecipitation of endogenous syntaxin 7 and VAMP7 from VacA-treated or nontreated AGS cells followed by immunoblotting. (A) AGS cells were transfected with the GFP-tagged syntaxin 7 vector and incubated for 24 h with or without VacA. The cellular extracts were immunoprecipitated using an anti-GFP polyclonal antibody, and immunoblotting was performed using an anti-GFP monoclonal antibody and anti-VAMP7 antibody. Since SNARE proteins can be sticky, the membranes were also immunoblotted for syntaxin 4, syntaxin 6, VAMP2, and VAMP8 to prove that the interaction between VAMP7 and syntaxin 7 was not nonspecific. (B) AGS cells were transfected with the GFP-TiVAMP/VAMP7 vector and incubated for 24 h with or without VacA. The cellular extracts were immunoprecipitated using an anti-GFP polyclonal antibody, and immunoblotting was performed. The total cell lysate (TCL) of AGS cells was used as a control for the bands. When the samples of immunoprecipitation from cells not expressing GFP were immunoblotted for VAMP7 and syntaxin 7, we could not detect the specific bands (data not shown). The experiments were repeated three times independently, and representative figures are shown.