Protein Extraction and Western Blotting

PTECs were lysed in RIPA buffer [50 mM Tris·HCl, pH 7.3; 150 mM NaCl; 0.1 mM EDTA; 1% (vol/vol) sodium deoxycholate; 1% (vol/vol) Triton X-100; 0.2% NaF; and 100 mM Na3VO4] supplemented with complete protease inhibitors (Roche Applied Science, Indianapolis, IN). The kidney homogenate was centrifuged at 12,000 × g for 30 minutes at 4 °C, and the protein concentration of the supernatant was determined by the Bradford method. Different amounts of extracted protein were separated by SDS-PAGE and transferred onto Immobilon-FL 0.4-μm polyvinylidene difluoride membranes (Millipore). Tris-buffered saline containing 0.1% Tween 20 was used as the washing buffer. After probing with primary antibodies, anti-rabbit (1:5,000; Cell Signaling Technology, Danvers, MA) and anti-mouse (1:6,000 for β-actin; Cell Signaling Technology) antibodies were used as secondary antibodies. Detection of labeled proteins was performed with an enhanced chemiluminescence system (ECLTM PRN 2106; Amersham Pharmacia Biotech, Buckinghamshire, UK). The band intensities were analyzed using a gel documentation system (Bio-Rad Gel Doc 1000 and Multi-Analyst version 1.1)^{17 (link)}.
Western immunoblotting was performed using primary antibodies against collagen 1 (Abcam), fibronectin (Santa Cruz Biotechnology, Dallas, TX), αSMA (Abcam), and β-actin (Cell Signaling Technology).