Renal Protein Quantification by Western Blot

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.