Western blot analysis

The cells were washed twice with PBS at 4°C and lysed on ice in RIPA lysis buffer containing 50 mM tris(hydroxymethyl) aminomethane (Tris)–HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 0.25% Na-deoxycholate, 1 mM Na₃VO₄, and 1 mM NaF and 2 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 10 µg/mL pepstatin, and 10 µg/mL leupeptin. The lysates were centrifuged at 12,000 rpm for 20 min at 4°C, and then the supernatants were collected for bicinchoninic acid protein assay. Total proteins (30 µg) were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membrane. The membranes were incubated with primary antibodies for cleaved PARP, Bcl-2, Mcl-1, Bcl-xl, c-FLIP, CIAP1, XIAP, p53, NQO1, and β-actin, which were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), at 4°C overnight, washed, and then incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) at 37°C for 1 h. The membranes were washed and detected using enhanced chemiluminescence detection system according to the manufacturer’s protocols.