Smad7

Total proteins were extracted from liver tissue and cells using radio-immunoprecipitation buffer (RIPA). 80 μg of sample proteins were separated by in 10 or 12% SDS-PAGE gel, and transferred onto PVDF membranes (Millipore Corporation, Billerica, MA, USA) by electrobloting. The membranes were blocked for 60 min in a buffer containing 0.1% Tween-20 and 5% milk. The membranes were incubated overnight at 4 °C with primary antibodies against α-SMA, Col-1, MMP-2 (Bioss, Beijing, China), Smad7 (Novus Biologicals, Littleton, USA), Wnt1 (Abcam, Cambridge, MA, USA), Wnt5a (Novus Biologicals, Littleton, USA), β-catenin, GSK-3β (ProteinTech Group, Chicago, USA), NFAT5 (Santa Cruz, CA, USA). Immune complexes were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies (ProteinTech Group, Chicago, USA), and then were visualized by ECL method. β-actin (Boster, Wuhan, China) was served as a loading control. Intensity of each protein band of interest was quantified by densitometry using Quantity One 4.6.3 software (Bio Rad).