Smad2 3

Total protein was extracted from liver tissue or HSCs, then washed with ice-cold PBS and lysed with RIPA Cell Lysis Buffer (Cell Signaling) containing a phosphatase inhibitor and the protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), by incubating on ice for 30 min. Lysates were collected by centrifugation and protein concentrations were determined by the Bicinchoninic Acid (BCA) method. The samples (30 μg protein/lane) were separated on 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. After blocking in Tris Buffered Saline (TBS) buffer (50 mmol/l NaCl, 10 mmol/l Tris, pH 7.4) containing 5% non-fat milk, the blots were incubated with primary antibodies (anti Periostin, anticollagen, anti-α-SMA, anti-p-Smad2, anti-p-Smad3, Smad2/3 or β-actin; Invitrogen) at 4°C overnight. Membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies. The blots were visualized by super ECL Western Blotting Substrate Kit and quantified by the Quantity ONE (Bio-Rad, Hercules, CA, USA) software. β-actin was used as the internal control.

TGF-β1, Smad2/3, MMP-2, MMP-9, TIMP-1, Collagen I, Collagen III, and Collagen IV primers were synthesized by Invitrogen (Grand Island, NY, USA). TRIzol reagent, PrimeScript™ RT reagent Kit, and SYBR Premix Ex Taq II Kit were provided by Takara Biotechnology Co., Ltd. (Dalian, Liaoning, China). TGF-β1, Smad2/3, P-Smad2/3, Smad4, MMP-2, MMP-9, GAPDH, and anti-rabbit secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All other chemicals and reagents were obtained from Sigma Chemicals (St. Louis, MO, USA).