Quantitative Western Blotting of Mitochondrial Dynamics

Western immunoblotting was performed as described previously [3 (link)]. Briefly, total protein samples were separated by SDS-PAGE and then transferred to PVDF membranes (Sigma, USA), which were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with the primary antibody (Nrf2, #GTX103322; Keap1, Novus, USA, #NBP1-83106; optic atrophy-1 (Opa1), ImmunoWay, USA, #YN2976; mitofusins2 (Mfn2), Abcam, USA, #ab124773; dynamin-related protein-1 (Drp1), Abcam, #ab56788; p-Drp1-Ser637, Cell Signaling Technology, USA, #4867; fission 1 (Fis1), GeneTex, #GTX111010; and GAPDH, Proteintech, China, #600004-1). HRP-labeled goat anti-rabbit/mouse IgG (H+L) antibody (Antgene, China) was used as the secondary antibody. The proteins were detected by a Bio-Rad imaging system.