Tead1

Cells were harvested and resuspended in radioimmunoprecipitation assay (RIPA) buffer, then incubated on ice for 20 min. Whole cell extract concentration was measured by Bradford assay (Pierce, 23236). Proteins were balanced and subject to SDS-PAGE analysis. Primary antibodies used were TEAD1 (1:1000, Cell Signaling Technology, 12292S) and beta-actin (1:1000, Cell Signaling Technology, 4970). The membrane was imaged using the Thermo Fisher iBright Imaging System.

For immunoprecipitation assay, artery homogenates or 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). Samples were then incubated with primary antibody at 4°C overnight, and immunocomplexes were precipitated after 1 hour of incubation with sepharose A/G beads (Santa Cruz, Delaware, CA). Western blot analysis was performed as described previously [46 (link)]. Extraction of artery and VSMC were lysed with RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). Antibodies against phospho (p)-NF2^Ser518, NF2, p-YAP^Ser127, YAP, TEAD1, GAPDH, H3, β-actin were purchased from Cell Signaling Technology (Danvers, MA). The luminescent signal was determined by exposure to x-ray film and quantitative analysis was completed with Image J software (Bethesda, MA).