Western Blot Analysis of Helicobacter Virulence Factors

For Western blotting, total protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Atlanta, GA, USA) [17 (link)]. After washing and blocking, the membranes were incubated with primary antibodies against VacA (sc-32,746, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:500), p65 NF-κB (ab32536, 1:5000), p-p65 NF-κB (ab76302, 1:1000), and GAPDH (ab8245; Abcam, 1:10,000). After washing again, the membranes were incubated with secondary antibody (ab205718 and ab6728; Abcam, 1:10,000) for 2 h at 25°C. Protein bands were detected using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA).