Genegenome system

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.

VICs were collected with radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific) supplemented with Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) and total protein concentration was determined (Thermo Fisher Scientific). Immunoblotting was performed as previously described.³⁸ Equal amounts of protein lysates were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% (v/v) skimmed milk in Phosphate Buffered Saline Tween‐20 (PBST), membranes were incubated overnight at 4 °C with primary antibodies (Table S3) diluted in 5% skimmed milk. Subsequently, membranes were incubated with HRP‐conjugated anti‐mouse (1:1000, P0047, Dako) or anti‐rabbit (1:1000, P0048, Dako) secondary antibodies at RT for 1 h. Membranes were developed using the GeneGenome system (Syngene). Semi‐quantitative assessment of band intensity was performed using ImageJ analysis software (National Institutes of Health).