2.10. Immunoblotting and immunoprecipitation

After washing with cold PBS, the cells were lysed in a lysis buffer (20 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA, 0.5% NP‐40, 10% glycerol, protease inhibitor cocktail [Roche]). Protein G beads coupled with 2 μg BMI1 antibody were incubated with cell lysates at 4°C for 4 h and then the beads were washed 4 times with lysis buffer. Proteins bound to the beads were separated using SDS‐PAGE and immunoblotted with anti‐BMI1 and DEFA5 antibody. The SGC7901 cells WT, BMI1^−/− and DEFA5 overexpression cells were washed twice with cold PBS and collected in lysis buffer. Proteins were separated using SDS‐PAGE and immunoblotted with anti‐AKT, p‐AKT, Erk1/2, p‐Erk1/2, p16, p19, β‐actin, BMI1 and DEFA5 antibodies.