2.11 Western blot analysis
Protein was extracted from tissues or cells by using RIPA lysate (Hat Biotechnology, China). After quantification with bicinchoninic acid, equal amounts of protein were loaded and separated by SDS-PAGE, then transferred electrophoretically to polyvinylidene difluoride membranes, which were incubated with primary antibodies overnight at 4℃, followed by a 1:5000 dilution of goat anti-rabbit IgG antibody (EK020, Zhuangzhi Biotech, China) at room temperature. Primary antibodies were for CTGF (1:1000, ab6992), Smad3 (1:1000, ab40854), Smad4 (1:1000, ab40759), p-Smad3 (1:500, ab193297), cyclin D2 (1:1000, ab230883), p21 (1:500, ab109199; all Abcam, England); EZH2 (1:500, 5246; CST, USA); Col I (1:500, WL0088), Col III (1:500, WL0318), β-actin (1:500, WL01372; all Wanleibio, China), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:1000, AF7021, Affinity Biosciences, USA). Protein bands were visualized by using a potent ECL kit (KF001, Affinity Biosciences, USA) and the Gene Company imaging system (China). GAPDH or β-actin was an internal reference.