VSMC Protein Extraction and Analysis

Carotid arteries and VSMCs were extracted, lysed with RIPA buffer (Beyotime, Shanghai, China), and then subjected to western blotting analysis as described in our previous research [5 (link)]. VSMC extracts were prepared in lysis buffer (50 mmol/L Tris·HCl (pH 7.5), 150 mmol/L NaCl, 0.5% IGEPAL CA-630, 0.5% deoxycholic acid, 0.1% SDS, 1 mmol/L EDTA, 1 mmol/L NaF, 0.1 mmol/L Na₃VO₄, 50 μmol/L phenylmethylsulfonyl fluoride, 5 μg/mL leupeptin, 5 μg/mL aprotinin; Solarbio, Beijing, China) for immunoprecipitation assay. Samples were incubated with the primary antibody at 4°C overnight, and immunocomplexes were precipitated after 1 h of incubation with sepharose A/G beads (Santa Cruz, Dallas, USA). Antibodies against PGC-1α (cat ab106814) and GAPDH (cat 2118S) were purchased from Abcam and Cell Signaling Technology, respectively. Antibody against Plin5 was purchased from Novus (cat NB110-60,509).