Anti smad2

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.

After 48-72 hours of relevant treatment, the cultured cells were washed twice with PBS (phosphate-buffered solution), and then, 50-100 µl RIPA lysis buffer (Sangon Biotech, Shanghai, China) supplemented with PMSF (phenylmethane sulfonyl fluoride), phosphatase and protease inhibitor (KeyGEN BIOTECH, Nanking, China) was used to elute the total protein. After measurement of protein concentration with a BCA (bicinchoninic acid) kit (Merck Millipore, Darmstadt, Germany), 50-100 µg of total protein was loaded and separated on a 10% SDS polyacrylamide gel, transferred to a PVDF (polyvinylidene difluoride) membranes, and incubated with anti-p21, anti-p27, anti-CDK2, anti-phospho-c-Jun or anti-phospho-Smad2 monoclonal rabbit antibody (1:1000, CST, Danvers, MA, USA); anti-E-cadherin, anti-β-catenin, anti-vimentin, anti-snail, anti-slug, anti-c-Jun, anti-Smad2, anti-CREB1, anti-JNK1 or anti-GAPDH rabbit polyclonal antibody (1:1000-1:5000, Proteintech, Chicago, IL, USA); or anti-CCAR1 rabbit polyclonal antibody (1:1000, SAB, Baltimore, MD, USA) overnight at 4°C. Protein bands were visualized using a chemiluminescence kit (Merck Millipore, Darmstadt, Germany).