Western Blot Analysis of Apoptosis Markers

Western blot analysis was performed as previously described [24 (link)]. Cells were lysed in RIPA lysis buffer (Beyotime, China) on ice and total protein was quantified by a bicinchoninic acid assay. Equal amounts of protein samples (40 µg) were loaded onto each lane of 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were incubated with the primary antibodies at 4 °C overnight following blocking with 5 % non-fat milk for 1 h. Then, the membranes were incubated with secondary antibodies for 1 h. Finally, signals were visualized with enhanced chemiluminescence reagent (Bio-Rad, USA), and the intensity of band was quantified using image software (Bio-Rad, USA). The antibodies against HMGA1, cleaved-caspase-3, bax, bcl-2, CHOP, GRP78 and β-actin were purchased from Invitrogen. β-actin was chosen as an internal control.