Anti smad3 antibody

Western blot was performed according to previous studies [24 (link), 32 (link)] to assess inflammation (COX-2) and collagen deposition (α-SMA, MMP-9) to further delineate the mechanism of emodin (SMAD3). Mammalian protein lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) was used to extract the total tissue protein. The same amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated in the diluted primary antibodies overnight and stored at 4°C. The primary antibodies were anti-COX-2 antibody (Santa Cruz, 1 : 200 dilution), anti-SMAD3 antibody (Santa Cruz, 1 : 500 dilution), anti-α-SMA antibody (Santa Cruz, 1 : 200 dilution), and anti-beta-actin (β-actin) antibody (Santa Cruz, 1 : 1000 dilution). The membranes were incubated with the secondary antibody followed by horseradish peroxidase (HRP; Santa Cruz). Then, an enhanced chemiluminescence system (EMD Millipore) was used to detect the bands. The intensity of the bands was calculated using Image-Pro Plus 5.0 software (Media Cybernetics Inc., Rockville, MD, USA).

ChIP was carried out using an anti-Smad3 antibody (Santa Cruz Biotechnology) or mouse IgG (Millipore, Billerica, MA, USA) and chromatin extracts equivalent to 2 × 10 cells. ChIP samples were quantified by qPCR (SYBR Green Master Mix: Applied Biosystems) and the ChIP qPCR data were normalized using the percent input method. The primer were listed in Supplementary Table S1.