Protein Extraction and Western Blot Analysis

According to the previously described method [19 (link)], total protein extraction was applied with RIPA buffer, adding a protease inhibitor cocktail, phenylmethylsulfonyl fluoride, and phosphatase inhibitors. Protein concentration was measured using a bicinchoninic acid protein assay kit (Wuhan Servicebio Technology, Ltd., China). Equal amounts of protein were loaded into the wells of SDS-PAGE (10%) and separated. A semidry transfer printer (Bio-rad, United States) was used to transfer protein from gel to PVDF membranes. Nonfat milk (5%) was used to block nonspecific epitopes for 90 min. The membranes were incubated with primary antibodies for α-SMA, TGF-β1, Collagen I, Collagen III, SOX9, Smad3, phospho-Smad3, and GAPDH according to instructions overnight at 4°C. After washing three times with TBST, the membranes were incubated by the corresponding secondary antibodies labelled with HRP for 90 min at RT. The blot bands were visualised by the ECL (Invitrogen) reagents and Fusion FX5 Spectra (VilberLourmat, France). Each protein was quantified by transmittance densitometry using Image J 3.0 software (National Institute of Health).