Protein Expression Analysis by Western Blotting

Western blotting was performed as previously described (Wang et al., 2015 (link)). Briefly, cell protein extracts (15–20 μg) were separated by 10–15% SDS-PAGE and transferred to PVDF membranes. Proteins were detected with the following antibodies: SIRT3 (1:1000, Cell Signaling Technology, Boston, MA, USA), MnSOD (1:1000, Proteintech, Chicago, IL, USA), cyclophilin D (CypD; 1:1000, Abcam, Cambridge, UK), cytochrome C (Cyt C; 1:1000, Cell Signaling Technology), cleaved caspase-3 (1:1000, Cell Signaling Technology), Bax (1:1000, Cell Signaling Technology, Boston, MA, USA), B-cell lymphoma 2 (Bcl-2; 1:1000, Cell Signaling Technology, Boston, MA, USA), COX-IV (1:1000, Abcam, Cambridge, MA, USA) and GAPDH (1:2000, Proteintech, Chicago, IL, USA).