Western Blot Analysis of Apoptosis Markers

Western blot analysis was performed as previous described [29 (link)]. Briefly, total protein was extracted from the cells using RIPA lysis buffer (Cwbio, Beijing, China), which was added with Protease Inhibitor Cocktail (1%, v/v) (Cwbio, Beijing, China). Then proteins were separated by 8% and 12% SDS-PAGE gels and electrotransfer onto Immobilon PVDF membranes (Merck KGaA, Darmstadt, Germany). After blocking the membranes with fat free milk, the membranes were incubated overnight at 4 °C with primary antibodies (Table 2). After incubation with secondary antibodies (goat anti-rabbit IgG, Proteintech, China), membranes were then imaged by an enhanced chemiluminescent detection kit (Cwbio, Beijing, China).Table 2

Antibodies used for Western blotting

Name	Description	Manufacturer
Anti-β-actin	Rabbit monoclonal, 43 kDa	Proteintech (20,536–1-AP)
Anti-Caspase-3	Rabbit monoclonal, 34 kDa	CST (#9662S)
Anti-Cleaved caspase-3	Rabbit monoclonal, 16 kDa	CST (#9664S)
Anti-CHD8	Rabbit monoclonal, 290 kDa	CST (#77,694)
Anti-p21	Rabbit monoclonal, 21 kDa	Abcam (109,199)