Psmad2 3 ser423 425

Whole cell lysate (40–50 μg proteins) from each sample was mixed with sample loading buffer and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were electrotransferred onto a polyvinylidene fluoride membrane, and blocked with 1x Tris-buffered saline-Tween 20 (TBST) containing 5% fat-free milk for 1 h at room temperature and then incubated overnight at 4°C in TBST containing 3% fat-free milk with primary antibodies (1:250 dilution). The membrane was treated with corresponding secondary anti-mouse or anti-chicken horseradish peroxidase-conjugated antibodies (1:5000 dilutions). Protein bands were developed using a SuperSignal West Femto Chemiluminescent kit and visualized using an Alpha Innotech detection system from Proteinsimple (Santa Clara, CA, USA). The intensity of protein bands was quantified by Alphaview software. The primary antibodies; δEF1 (catalog # sc-10573), pSmad2/3 (Ser423/425; catalog # sc-11769), H1 (catalog # sc-10806), α-SMA (catalog # A-7607), COL1A2 (catalog # sc-8788), and β-actin (catalog # A5316) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Primary antibody for NPRA was produced as previously described [16 (link), 65 ].