Cyclophilin d

Cardiomyocyte LVFW samples were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) supplemented with protease inhibitor cocktail and centrifuged at 10,000 × g for 15 min. Protein content of samples was determined by the Bradford assay (Bio-Rad Laboratories, Carlsbad, CA, USA). Aliquots of each sample were separated in a 12% Tris/glycine sodium dodecyl sulfate/polyacrylamide gel and then transferred onto a nitrocellulose membrane that was blocked with skimmed milk for 1 h. The membranes were respectively probed overnight at 4°C with antibodies against the following proteins: Bcl-2 (1:1,000 dilution), Bax (1:1,000 dilution), cytochrome c (1:500 dilution), caspase-9 (1:1,000 dilution), caspase-3 (1:500 dilution), PARP (1:1,000 dilution), and ANT (1:200 dilution) (Cell Signaling Technology); β-actin (1:2,000 dilution), TOM20 (1:500 dilution), and VDAC (1:500 dilution) (Santa Cruz); and cyclophilin D (1:100 dilution; Millipore). After washing with Tris-buffered saline-Tween (TBS-T) buffer three times, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Beyotime Institute of Biotechnology) for 1 h, and the bound proteins were detected with an enhanced chemiluminescence kit (Thermo Fisher Scientific). The blots were scanned and quantified using ImageJ program (NIH, Bethesda, MD, USA).