Horseradish peroxidase conjugated secondary antibodies

Renal protein extracts were obtained using ice-cold lysis buffer (Cell Signaling) supplemented with protease and phosphatase inhibitors (Cell Signaling). Equal amounts of protein were subjected to electrophoresis in SDS-polyacrylamide gel under reducing conditions and blotted to nitrocellulose membrane using the Bio-Rad Transblot Turbo Transfer System (Hercules, CA, USA). The membranes were blocked in 5% BSA, 0.1% Tween-20 in Tris-buffered saline (TBST) for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies (1:1000). Membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (R&D Systems, Minneapolis, MN, USA) for 1 hour at room temperature; immune complexes were detected with the enhanced chemiluminescence ECL detection system (Bio-Rad) in the ImageQuant LAS 4000 biomolecular imaging system (GE Healthcare, Pittsburg, PA, USA). The intensity of bands for each protein was normalized to the loading control alpha-tubulin (Cell Signaling, #A2148). The specific primary antibodies used for immunoblotting were: anti-fibroblast-specific protein 1 (aka S100A4, Cell Signaling, #13018); anti-platelet-derived growth factor receptor beta (PDGFRβ, Sigma-Aldrich, #06–498-I); anti-snail (Cell Signaling, #3879); and anti-vimentin (Sigma-Aldrich, #V2258).