Ab32023

Renal tissues and cells were lysed using RIPA lysis fluid. The protein concentration was determined using a BCA protein assay kit (P0010S, Beyotime, China). Equal amounts of protein were subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk and incubated with one of the following primary antibodies: anti-Cleaved caspase-3 (9661/R, 1:1000, Cell Signaling Technology, United States), anti-SIRT1(ab189494, 1:1000, Abcam, UK), anti-PGC-1α(ab191838, 1:1000, Abcam, UK), anti-NRF1(ab175932, 1:1000, Abcam, UK), anti-TFAM(A13552, 1:1000, Abclonal, China), anti-MFN1(13798-1-AP, 1:1000, Proteintech, China), anti-DRP1 (ab184247, 1:1000, Abcam, UK), anti-p66Shc (ab33770, 1:1000, Abcam, UK), anti-cytochrome C(CytoC) (ab133504, 1:5000, Abcam, UK), anti- DIABLO (ab32023, 1:1000, Abcam, UK), overnight at 4°C. After washing with TBST, the PVDF membranes were incubated with a secondary antibody at 37°C for 1 h. The protein bands on the PVDF membranes were observed.

Proteins were extracted from the primary cardiomyocytes in RIPA buffer (1% Triton X-100, 150 mmol/L NaCl, 5 mmol/L EDTA, and 10 mmol/L Tris-HCl, pH 7.0; Solarbio, China) supplemented with a protease inhibitor cocktail (Cat: I3786-1ML, Sigma). The cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to a PVDF membrane (Millipore Corporation, USA). After blocking with 8% milk in PBS, pH 7.5, the membranes were incubated with the following specific primary antibodies of Bax (ab32503), Bcl-2 (ab59348), cytochrome C (ab13575), Smac/Diablo (ab32023), cleaved-capase-3 (ab13847), cleaved-capase-9 (ab2324), p-p38 (ab47363), p38 (ab31828), p-ERK (ab214362), and ERK1/2 (ab196883; all at a dilution of 1:1000, Abcam, UK). After overnight incubation, the appropriate HRP-conjugated anti-rabbit IgG secondary antibody (ab205781, Abcam, all at a dilution of 1:5000) was subsequently applied and immunodetection was achieved using the ECL Plus detection system (Millipore Corporation) according to the manufacturer's instructions. Band intensity was quantified using Image Lab™ Software (Bio-Rad, China). GAPDH (ab8245, Abcam) was used as an internal control.