Anti smad7

Western blot analyses of HCF and HKC cells were performed with lysis of cells, as previously described [27 (link),31 (link),32 ]. Protein concentration and purity were assessed by Bradford assay (Thermo Scientific, IL). As well, 4%–20% Tris-Glycine gels (Novex, Life technologies, Carlsbad, CA) was used for gel electrophoresis, to which equal amounts of proteins were loaded and a protein transfer was done using Nitrocellulose membrane (Novex, Nitrocellulose membrane filter par sandwich, Life Technologies). After incubation in a 5% BSA blocking solution (Thermo Scientific), the membranes were incubated with primary rabbit antibodies (Table 2): anti-Smad3 (Invitrogen, Camarillo, CA), anti-SMAD7 (Sigma-Aldrich, Saint Louis, MO), anti-SMAD4 (Abcam, Cambridge, MA), anti-SMAD6 (Abcam), Anti-TGF-βRI (Abcam), Anti-TGF-βRII (Abcam), Anti-TGF-βRIII (Abcam), anti-TGF-β1 (Abcam), anti-TGF-β2, and anti-TGF β3 (Abcam) at 1:1,000 dilution overnight at 4 °C separately. This was followed by washing of the membranes and incubation with a secondary antibody (Alexa Flour® 568 Donkey anti-Rabbit, IgG [H⁺L], Abcam) at 1:2,000 dilutions for 1 h. The Kodak imaging system was used for detecting the antibody binding to the membrane. GAPDH (Abcam) was used as the loading control and results were analyzed by normalizing the value to that of the loading control expression and plotting the fold expression.