Rabbit anti smad2 3 antibody

Mouse anti-β-actin monoclonal antibody was obtained from Sigma (St. Louis, MO, USA). Rabbit polyclonal anti-TGF-β, rabbit polyclonal anti-α-smooth muscle actin (SMA), horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG), rabbit polyclonal anti-type IV collagen, rabbit polyclonal anti-TGF-β, rabbit anti-Smad2/3 antibody, rabbit anti-pSmad2/3 antibody and horseradish peroxidase-labeled goat anti-rabbit IgG were purchased from Abcam (Cambridge, MA, USA). The spare kidney tissue was removed and thawed and homogenized for radio immunoprecipitation. The homogenate was centrifuged for 10 minutes at 12 000 rpm in a 4°C cryogenic high-speed centrifuge, and then, the supernatant was collected. Quantitative 70 μg protein samples were prepared by gel electrophoresis and then transferred to a cellulose acetate membrane. A 1x casein solution was used to block the membranes for 1 hour after transfer. After blocking, the membrane was transferred to the refrigerator and stored at 4°C and incubated overnight with the first antibody. Then, the samples were washed thoroughly and incubated with the second antibody. Images of protein imprinting were analyzed using Image J 1.42 software after antibody incubation.

Besides the 10 BAVM samples for iTRAQ analysis, another 6 BAVM samples were also collected for further validation. Equal amounts of protein were separated by SDS-PAGE and then electro-transferred to PVDF membranes. Subsequently, membranes were blocked with the PBST containing 5% bovine serum album (Sigma-Aldrich, United States) for 1 h and then probed with primary antibodies at 4°C overnight, including rabbit anti-DCN antibody (Abcam, United Kingdom) at 1:1000, rabbit anti-Smad2/3 antibody (Abcam, United Kingdom) at 1:100, rabbit anti-Col I antibody (Abcam, United Kingdom) at 1:1000, and rabbit anti-GAPDH antibody (Abclonal Technology, Wuhan, China). After primary incubation and being washed with PBST, membranes were incubated with secondary antibodies at 1:5000 for 1 h at room temperature. Bands were visualized by an ECL detection system (GeneSys, Alcatel, France). The expression levels were quantified with ImageJ (version 1.8.0, a free software). The expression levels of target proteins were evaluated by performing densitometric analysis. Ratios of target protein densitometric measurements to GAPDH were used for further analysis.