Active β catenin clone 8e7

The following antibodies were used for immunofluorescence (IF) and/or Western blot analysis: SMI31 – 1:1000 for WB and IF (Millipore, NE1022). MBP – 1:500 for WB and IF (Millipore, MAB382). β-catenin WB 1:500 (BD Biosciences, 610153). Active β-catenin (clone 8E7) (Millipore). α-bungarotoxin (Btx: to visualise NMJ, Invitrogen). F69-3G10 – 1:500 for WB (referred to as 3G10 in results) (Amsbio). F58-10E4 – 1:100 for IF (referred to as 10EA in results)(Amsbio). Sox2 – 1:1000 for Western blot (Abcam). WB secondary: Mouse IgG HRP-linked (GE Healthcare, NXA931). IF secondary: Alexa Fluor mouse IgG1, IgG2a (ThermoFischer Scientific, A-21121).

Proteins were extracted from whole kidneys sections and were homogenized in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with protease and phosphatase inhibitors mixture. Proteins were separated by 10–12% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked using 5% skim milk or BSA in 0.1% PBS-Tween for 1 hour at room temperature. The immunoblotting was performed using primary antibody against total β-catenin (# sc-7963, Santa Cruz Biotechnology, Santa Cruz, CA), active β-catenin clone 8E7 (# P35222, Millipore, Billerica, MA), total GSK-3β (# sc-9166, Santa Cruz Biotechnology, Santa Cruz, CA), p-Ser9-GSK-3β (# 93365, Cell Signaling, Danvers, MA), and the target genes cyclin D1, c-myc, and bcl-2 (# sc-717, sc-788, and sc-7382, respectively, Santa Cruz Biotechnology, Santa Cruz, CA) or fibronectin (# F3648, Sigma Aldrich, St. Louis, MO) and incubated overnight at 4°C. Proteins were detected using enhanced chemiluminescence techniques. The β-actin levels (# A1978, Sigma Aldrich, St. Louis, MO) were used as a load control and the densitometric analysis was performed using ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD).