Goat anti mouse or anti rabbit hrp conjugated secondary antibody

Cells were lysed and total protein extracted in RIPA lysis buffer (Applygen, Beijing, China) supplemented with protease inhibitors (Roche, Indianapolis, IN) and phosphatase inhibitors (Applygen, Beijing, China) at 4 °C for 30 min. The cell lysate was centrifuged at 12,500 rpm at 129,50 g-force for 15 min at 4 °C and protein concentrations were measured by BCA method (Beyotime, Guangzhou, China). Proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA). After blocking with 5% fat-free milk in TBST for 1 h, membranes were immunoblotted with primary antibodies to caspase3, cleaved-caspase3, PARP, cleaved-PARP, p21, p-RB, PLK1, p-CDK1, and Aurora B (Cell Signaling Technology); p53, FOXM1, Aurora A, CDC-25C, CDK-1, cyclin B1, TOP2A, and GAPDH (Proteintech) at the appropriate dilutions with gentle shaking overnight at 4 °C. Blots were incubated with goat anti-mouse or anti-rabbit HRP-conjugated secondary antibody (1:3000; CWBIO, Beijing, China) and bands were visualized using an enhanced chemiluminescence, eECL Western Blot Kit (CWBIO, Beijing, China).