Atp5d

Rg1 was obtained from Feng Shan Jian Medicine Research Co. Ltd. (Kunming, China). Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) were purchased from Sigma (St. Louis, MO, USA). ELISA kits for ATP, ADP, AMP, and cTnI were from Beijing Andihuatai Technology Co. Ltd. (Beijing, China). The enzyme activity assay kits of complex I, complex II, and ATP synthase, the primary antibodies against cTnI, ROCK1, phospho-RhoA, RhoA, phospho-MYPT1, human RhoA full-length protein, and ATP5D were obtained from Abcam (Cambridge, MA, USA). The primary antibodies against Bax, Bcl-2, cleaved-caspase-3, MYPT1, and GAPDH were obtained from Cell Signaling Technology (Beverly, MA, USA).

The mixture of whole protein and 5× electrophoresis sample buffer was subjected to electrophoresis on 10 or 12% SDS-PAGE. The separated proteins were transferred to a polyvinylidene difluoride membrane. Following blocking with 5% defatted milk powder, rinsing with TBS-Tween, the membrane was cut and incubated overnight at 4°C with antibodies against GAPDH (1:4,000, CST, VT, United States), Caspase 3 (1:1,000, CST, VT, United States), cleaved Caspase 3 (1:1,000, CST, VT, United States), Caspase 9 (1:1,000, CST, VT, United States), cleaved Caspase 9 (1:1,000, CST, VT, United States), Bcl2 (1:1,000, CST, VT, United States), Bax (1:2,000, CST, VT, United States), ATP 5D (1:1,000, Abcam, Cambridge, United Kingdom), RhoA (1:1,000, Abcam, Cambridge, United Kingdom), ROCK-1 (1:1,000, Abcam, Cambridge, United Kingdom), phosphorylated myosin light chain (P-MLC) (1:1,000, CST, VT, United States), Sirt-1 (1:800, CST, VT, United States), and NDUFA10 (1:2,000, Santa Cruz, CA, United States). After rinsing, the membrane was incubated with HRP-conjugated secondary antibody (1:5,000, Cell Signaling Technology, VT, United States) for 1 h at room temperature. The target protein was quantified by scanning densitometry with a bio-image analysis system (Image-Pro plus 6.0; Media Cybernetic, Bethesda, MD, United States).