Immunoprecipitation and Western Blot Analysis

Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor tablet (Roche) as previously described (Li et al., 2019c (link); Lu Y. Y. et al., 2019 (link)). Specific antibodies or pre-immune IgGs (P.I.I.) were added to and incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads (Santa Cruz). Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Alternatively, FLAG-conjugated beads (M2, Sigma) were added to and incubated with lysates overnight. Precipitated immune complex was eluted with 3X FLAG peptide (Sigma). Western blot analyses were performed with anti-FLAG (Sigma, F1804), anti-V5 (Sigma, F3165), anti-β-actin (Sigma, A2228), anti-BRG1 (Santa Cruz, sc-17796), anti-ETS1 (Cell Signaling Tech, 14069), anti-PR65α (Proteintech, 15882-1), anti-PR65β (Proteintech, 12621-1), anti-PP2Cα (Proteintech, 13482-1), anti-PP2Cβ (Proteintech, 12554-1), anti-eNOS (Santa Cruz, sc-654), and anti-eNOS-Ser1177 (Santa Cruz, sc-12972).