Cyclophilin d cypd

Western blotting was performed as previously described (Wang et al., 2015 (link)). Briefly, cell protein extracts (15–20 μg) were separated by 10–15% SDS-PAGE and transferred to PVDF membranes. Proteins were detected with the following antibodies: SIRT3 (1:1000, Cell Signaling Technology, Boston, MA, USA), MnSOD (1:1000, Proteintech, Chicago, IL, USA), cyclophilin D (CypD; 1:1000, Abcam, Cambridge, UK), cytochrome C (Cyt C; 1:1000, Cell Signaling Technology), cleaved caspase-3 (1:1000, Cell Signaling Technology), Bax (1:1000, Cell Signaling Technology, Boston, MA, USA), B-cell lymphoma 2 (Bcl-2; 1:1000, Cell Signaling Technology, Boston, MA, USA), COX-IV (1:1000, Abcam, Cambridge, MA, USA) and GAPDH (1:2000, Proteintech, Chicago, IL, USA).

The total proteins were extracted from rat mesenteric arteries or cultured HUVECs using RIPA lysis buffer containing Halt^TM protease and phosphatase inhibitors (78442, Thermo Fisher Scientific, Waltham, MA, USA). The protein concentrations were determined using a BCA protein assay (23225, Thermo Fisher Scientific). Equal amounts of protein from each sample were separated by 10 % SDS–PAGE and then transferred to PVDF membranes (IPVH00010, Millipore, Burlington, MA, USA). Following blocking with 5% BSA in TBS with Tween–20 for 60 min, the membranes were probed with primary antibodies at 4 °C overnight. After being rinsed by TBST, the membranes were further incubated with peroxidase labeled secondary antibody diluted 1:5000 for 60 min at room temperature. The bands were detected by adding chemiluminescent HRP Substrate (Millipore, USA) and quantified using Image J software. The primary antibodies used were specific for p-eNOS (1:500 dilution; Cell Signaling Technology, Danvers, MA, USA), p-AKT (1:500 dilution; Cell Signaling Technology), AKT (1:500 dilution; Cell Signaling Technology, Danvers, MA, USA), eNOS (1:500 dilution; Cell Signaling Technology), BCL-2 (1:500 dilution; Abcam, Cambridge, UK), BAX (1:500 dilution; Abcam), cyclophilin D (CypD, 1:500 dilution, Abcam), OPA1 (1:1000 dilution, Abcam) and GAPDH (1:5000 dilution, Ray antibody Biotech, Peachtree Corners, GA, USA).