Immunoprecipitation Assay for NF2 and YAP Signaling

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).