Mitochondrial Protein Analysis in VSMCs

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer containing Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and total protein concentration measured using a commercially available BCA protein assay (Thermo Scientific). Mitochondrial fractions were isolated from cultured VSMCs using a commercial kit (89874; Thermo Scientific). Immunoblotting was performed as previously described [55 (link)]. Nitrocellulose membranes were incubated for up to 16 h at 4 °C with primary antibodies (1:1000 dilution in 5% milk in TBST), p53 (7F5; Cell Signaling Technology), p21 (2947; Cell Signaling Technology), TOM20 (DBT4N; Cell Signaling Technology), OPA1 (ab157457; Abcam, Cambridge, UK), DRP1 (ab184247; Abcam), DRP1-pS637 (BT-PHS00841; Bioassay Technology Laboratory, Birmingham, UK), TNAP (MAB 2909; R & D Systems, Minneapolis, MN, USA) and OCN (ab93876; Abcam). Membranes were then probed with HRP-conjugated goat anti-rabbit IgG (P0448; Dako) and goat anti-rat IgG (HAF005; Dako, Glostrup, Denmark) for 1 h. Membranes were developed using the GeneGenome system (Syngene, Frederick, MD, USA). Membranes were then washed and re-probed for mouse β-actin expression (1:10,000; A3845; Sigma). Semi-quantitative assessment of band intensity was performed using ImageJ image analysis software.