Protein Isolation and Western Blot Analysis

Protein isolation and Western blots were performed as previously described [68 (link)]. Briefly, cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS supplemented with protease inhibitors (Complete, Roche; 1 mM PMSF, Sigma-Aldrich; 1mM sodium orthovandate, Sigma-Aldrich). Total protein in the extracts was quantitated using a colorimetric BCA Protein Assay Kit as per the manufacturer's instructions. 40 μg of total cellular protein for each sample was boiled in Laemmli sample buffer and separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose membrane (Hybond-C Extra). Immunoreactive bands were visualized by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (containing equal parts of the Stable Peroxide Solution and the Luminol/Enhancer Solution) (Thermo Scientific, USA). Antibodies used for Western blotting were rabbit anti-PAX2 (1:500; Zymed), mouse anti-SMAD2/3 (1:1000; Santa Cruz Biotechnology), goat anti-GAPDH (1:2000, Santa Cruz Biotechnology), and anti-rabbit and anti-mouse horseradish peroxidase IgG (Sigma-Aldrich). The protein ladder used was MagicMark XP Standard (Invitrogen, USA).