Anti tgfbr1

The protein expressions of α-SMA, Col-1, TGFBR1, TGFBR2, Smad2 and Smad3 were determined using Western blot analysis. Total protein was extracted from liver tissues or cells using lysis buffer (Beyotime Biotechnology, Haimen, China) according to the manufacturer's instructions. The concentration of total protein was quantified using a BCA kit (Beyotime Biotechnology, Haimen, China) according to the manufacturer's instructions. The total protein (20μg per sample) was separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, Burlington, MA, USA), and the PVDF membranes were blocked with 5% skimmed milk for 1h. The proteins on PVDF membranes were then incubated with the primary antibodies of anti-α-SMA (1:2000, Abcam, Cambridge, UK), anti-Col-1 (1:1000, Novus Biologicals), anti-TGFBR1 (1:2000, Abcam, Cambridge, UK), anti-TGFBR2 (1:2000, Abcam, Cambridge, UK), anti-Smad2 (1:1000, Novus Biologicals, Littleton, CO, USA), anti-Smad3 (1:1000, Novus Biologicals, Littleton, CO, USA) and β-actin (1:2000, ProteinTech Group, Inc., Chicago, IL, USA) overnight at 4°C. The proteins on were then incubated with the appropriate HRP-conjugated secondary antibodies. The bands were visualized using ECL Substrate (Thermo Scientific, Waltham, MA, USA) and quantified using Quantity One imaging software (Bio-Rad, Hercules, CA, USA).