Protein Quantification and Western Blot Analysis

Cells were scraped with PBS and lysed with ice-cold RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitor cocktail. The lysates were centrifuged at 12,000× g for 15 minutes at 4°C, and the precipitate was discarded. Equal amount protein (10 µg) was electrophoresed with sodium dodecyl sulfate-polyacrylamide gel and then the proteins were transferred to a polyvinylidene difluoride membrane (Merck Millipore, Kenilworth, NJ, USA). The primary antibodies used in this study included anti-Sirt3 (Abcam, #ab86671), anti-Ku70 (Invitrogen, #MA5-13110), anti-acetyl (Abcam, #ab80178), anti-BAX (Cell Signaling Technology, Danvers, MA, USA; #2774), anticytochrome c (Cell Signaling Technology, #4280), anticaspase 3 (Cell Signaling Technology, #9662), and anti-β-actin (Cell Signaling Technology, #3700). After incubation with primary and secondary antibodies, the immunoreactivity bands were visualized by using an enhanced chemiluminescence substrate kit (Thermo Scientific, Pittsburgh, PA, USA).22 (link)