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.