2.6. Western Blot

Fresh-frozen distal stomach samples were grinded into cell suspension on ice using a homogenizer and homogenized in RIPA buffer with a protease inhibitor. After centrifugation at 12000 rpm for 5 min in 4°C, the supernatants were taken as the total protein. Loading buffer was mixed with the protein to scale prior to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The total protein was separated by 10% SDS-PAGE and then transferred to a PVDF membrane. According to molecular weight, these membranes were incubated with primary antibodies against c-kit (1 : 200, Invitrogen, USA), SCF (1 : 1000, R&D Systems, USA), NF-κB (1 : 2000, Proteintech, USA), p-NF-κB (1 : 1000, CST, Germany), IKK (1 : 600, Proteintech, USA), HO-1 (1 : 1000, Proteintech, USA), Nrf2 (1 : 5000, Proteintech, USA), and GAPDH (1 : 1000, Beyotime, China) at 4°C overnight. Then, secondary antibody was added for 2 h at 37°C. Signal detection was visualized using an enhanced chemiluminescent agent, and the blot was present and detected by autoradiography.