Goat anti rabbit

PSCs were harvested and then lysed with lysis buffer (1% cocktail of protease inhibitor and 1% phosphatase inhibitor in 1X RIPA buffer). The cells were boiled in sample buffer [62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 20% glycerol, and 10% 2-mercaptoethanol]. The protein concentrations were determined by bicinchoninic acid assay. Total cell proteins (20 µg) were separated via 10% SDS-polyacrylamide gel and transferred to a PVDF membrane (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). The membranes were blocked with 5% skim milk in PBS-Tween-20 (PBST) for 2 h at RT and incubated with primary antibodies (1:1,000) at 4°C overnight: α-SMA, collagen I, GAPDH, pSMAD2/3, pSMAD1/5, SMAD3, SMAD5. After washing thrice, the membranes were incubated with HRP-conjugated goat anti-mouse (1:5,000; cat. no. 1031-05; Southern Biotech, Birmingham, AL, USA) or goat anti-rabbit (1:5,000; cat. no. NB7156; Novus Biologicals, LLC, Littleton, CO, USA) IgG secondary antibodies for 1 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence detection system (Amersham; GE Healthcare), according to the manufacturer's recommended protocol. Capture of protein bands and quantitative analysis were performed using Quantity One^® software version 4,6,6 (Bio-Rad Laboratories, Inc, Hercules, CA, USA).