Pgc1 β e 9

Proteins were extracted from mouse AMVMs with ice‐cold modified radioimmunoprecipitation assaybuffer (P0013C, Beyotime Biotechnology, China) containing protease and phosphatase inhibitors. Lysates were centrifugated at 14 000 rcf for 15 minutes at 4 °C and protein lysates were transferred to a new Eppendorf tube for bovine serum albumen quantification. Protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 7.5% to 15% gradient gels (Bio‐Rad, USA) and transferred to polyvinylidene difluoride (IPVH00010, Millipore, Germany) membranes. The membranes were blocked for 2 hours with 5% DifcoTM Skim Milk (BD10610, BD Biosciences, USA) and incubated overnight at 4 °C with primary antibodies: Pgc1‐α (4A8) (1:1000, sc‐517380, Santa Cruz, USA), Pgc1‐β (E‐9) (1:1000, sc‐373771, Santa Cruz, USA), γ‐H2A.X (1:500, 9718S, CST, USA), histone H3 (1:1000, CY6587, Abways Technology, China), p53 (1:1000, 10442‐1‐AP, Proteintech, USA), DRP1 (D6C7) (1:1000, 8570S, Cell Signaling Technology, USA), and OPA1 (D6U6N) (1:1000, 80471S, Cell Signaling Technology, USA) followed by secondary antibody for 1 hour with a 1:10 000 dilution of IgG Goat Anti‐Mouse HRP (SA00001‐1, Proteintech, USA) or IgG Goat Anti‐Rabbit HRP (SA00001‐2, Proteintech, USA). The results were visualized using an Amersham Imager 600 (General Electric Company, USA).