Antibodies against cytochrome c

Lung tissues and cultured rat primary PASMCs were lysed in RIPA lysis buffer (Beyotime Inc, Jiangsu, China) containing 0.2 mmol/L phenylmethanesulfonyl fluoride. Lysate solutions were centrifuged at 12 000 rpm (×14000 g) for 20 minutes, and supernatant was collected. The protein concentrations were determined using a BCA protein assay kit (Beyotime Inc, Jiangsu, China). The protein suspensions of different groups, containing equal amounts of proteins (30 μg), were separated by SDS‐PAGE, transferred to polyvinylidene fluoride membrane (Millipore, Billerica, MA) and blocked with 5% milk solution. Membranes were incubated with the solutions of cleaved caspase3/poly‐ADP ribose polymerase (PARP), Bcl‐2, Bax, proliferating cell nuclear antigen, and COX‐IV (1:800, Cell Signaling Technology, Danvers, MA) and β‐actin (1ː2000, ImmunoWay, Plano, TX) at 4°C overnight. To measure the release of cytochrome C, the mitochondrial and cytosol pellets were isolated and immunoblotted by antibodies against cytochrome C (1:800, Cell Signaling Technology, Danvers, MA) with voltage‐deopendent anion channel (1:800; Cell Signaling Technology) and β‐actin serving as controls. Blots were then probed by an enhanced chemiluminescence reagent (Millipore, Billerica, MA) after incubation with the corresponding horseradish‐peroxidase‐conjugated antibody solution.