Whole-cell Lysate Extraction and Immunoprecipitation

Whole-cell lysates were obtained by resuspending cell pellets in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described (Dong et al., 2020 (link); Fan et al., 2020 (link); Li et al., 2020b ,c (link)). Specific antibodies or preimmune 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 1× SDS electrophoresis sample buffer. Western blot analyses were performed with anti-MKL1 (Proteintech, 21166-1), anti-KDM7A (Genetex, GTX32688), anti-SMAD2 (Proteintech, 12570-1), anti-SMAD3 (Abcam, ab208182), anti-SMAD4 (Proteintech, 10231-1), anti-RHOJ (Sigma, HPA003050), and anti-β-actin (Sigma, A2228) antibodies. All experiments were repeated three times.