Phospho nfκb p65 ser276

Mouse macrophage and THP-I whole-cell extracts were isolated for western blot analysis as described previously [37 (link)]. Proteins were resolved by SDS polyacrylamide gel electrophoresis (4–20%) and electrophoretically transferred to polyvinylidene fluoride membranes (0.2 μm pore size). Immunodetection was performed using chemiluminescent detection with the Renaissance Western Blot Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences Inc., Boston, MA). Blots were stripped using the Restore Western Blot Stripping Buffer (Pierce Biotechnology Inc., Rockford, IL) as described by the manufacturer. Purified rabbit polyclonal antibodies to phospho-NFκB p65 (Ser276, Cell Signaling), NFκB p65 (sc-109, Santa Cruz Biotechnology), iNOS (sc-650, Santa Cruz Biotechnology), and actin (sc-1616, Santa Cruz Biotechnology) were used. Optical densities of antibody-specific bands were determined using Quantity One acquisition and analysis software (Bio-Rad, Hercules, CA).

Total protein from renal cortical tissues were extracted by RIPA lysis buffe, and western blotting analysis was performed as previously described 15 (link)-18 (link), 20 (link)-22 (link), 24 (link). Antibodies used in this study included those against phospho-NF-κB/p65 (ser276) (Cell Signaling Technology, Berkeley, CA. cat# 3033), phospho-IκBα (ser32) (Cell Signaling Technology, cat# 2859), IκBα (Cell Signaling Technology, cat# 9242), phospho-Smad3 (Abcam, cat# ab52903), Smad3 (Invitrogen, cat# PA1-38613), Smad7 (Santa Cruz Biotechnology, cat# sc-9183), Smurf2 (Santa Cruz Biotechnology, cat# sc-2511), NF-κB/p65 (Cell Signaling Technology, cat# 8242), β-actin (Santa Cruz Biotechnology, cat# sc-47778), collagen I (Southern Biotech), FN (Abcam, cat# ab2413). After being incubated with the primary antibody at 4 °C overnight, the membrane was stained with the LI-COR IRDye 800-labeled secondary antibodies (1:3000, Rockland Immunochemicals, Gilbertsville PA, USA) for 1h. The signals were detected with Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NA, USA) and quantitated with Image J software (v1.48, NIH, USA). The intensity of the protein band was normalized against β-actin or total proteins as stated in the studies and expressed as the mean ± SEM.