B cell lymphoma 2 associated x bax

Protein levels were estimated by Western blotting assays. Total protein of cell lines was obtained with a RIPA kit (Beyotime, Shanghai, China). Using the bicinchoninicacid Protein Assay Kit (Beyotime, Shanghai, China), the protein concentration of each sample was measured. Denatured cellular protein lysates were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and electro-transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Membranes were blocked in 5% milk at room temperature for 1 h and incubated with primary antibodies overnight at 4°C. After three washes, membranes were incubated for 1 h at room temperature with goat anti-rabbit IgG or anti-mouse IgG (1:1000; Beyotime, Shanghai, China) as a secondary antibody. Protein bands were visualized using an enhanced chemiluminescent reagent (Millipore, Billerica, MA, USA). All primary antibodies, including DOK3 (1:1000), P65 (1:1000), phosphorylated-P65 (p-P65) (1:1000), B-cell lymphoma-2 like 11 (BIM) (1:1000), B-cell lymphoma-2 associated X (BAX) (1:1000), X-linked inhibitor of apoptosis (XIAP) (1:1000), and β-actin (1:1000) were purchased from Abcam (Abcam, Cambridge, England).