Bs 1165r

Total protein from the kidney tissue and NRK-52E cells was obtained with a protein extraction kit (Solarbio, Beijing, China) as directed by the manufacturer. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate proteins (50 μg/well). When bromophenol blue ran to the bottom of the gel, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. Blocking was carried out with 5% skim milk. The membranes were then washed with TBST three times for 10 min and subjected to incubation with the following antibodies overnight: β-catenin (1:500, bs-1165R, Bioss), Sfrp1 (1:500, bs-1303R, Bioss), p-GSK3β^ser9 (1:500, sc-373800, Santa Cruz), E-cadherin (1:500, bs-10009R, Bioss), and α-SMA (1:500, 55135-1-AP, Proteintech), collagen IV (col-IV) (1:1000, SAB4200500, Sigma), β-actin (1:4000, Lot:181620, Pumei), and GSK3β (1:500, sc-8257, Santa Cruz). The next day, the membranes underwent three TBST washes of 10 min. Secondary antibodies diluted with 1% skim milk were added to the membranes for 1 h at room temperature. Band intensities were assessed by Image Lab software.

Protein samples collection was performed using RIPA buffer (KeyGen, Shanghia, China) on ice for 15 min. Total protein contents were quantified by an Enhanced BCA Protein Assay Kit (Beyotime, Beijing, China). Aliquots of protein were loaded onto 10% SDS-PAGE gel, followed by transfer onto PVDF membranes (Millipore, Darmstadt, Germany). The membranes were subjected to blocking using 5% bovine serum albumin (BSA) for 1 h. The incubation with primary antibodies was performed at 4 °C overnight: cleaved caspase-3 (1:2000, Abcam, ab2302), Bcl-2 (1:2000; Abcam, ab59348), FZD4 (1:1500; Bioss, bs-13217R), β-catenin (1:1500; Bioss, bs-1165R), non-phospho (Active) β-catenin (1:1000; Cell Signaling Technologies, 8814), cyclin D1 (1:2000; Abcam, ab134175), c-Myc (1:1500; Abcam, ab190026), or GAPDH (1:5000; Bioss, bs-2188R). The next day, the membranes were washed 4 times with TBST buffer and then further labeled with HRP-linked secondary antibody (1:10000; Invitrogen, 61–6520) for 1 h at room temperature. After rinsing for 4 times signal development was conducted using a chemiluminescence kit (Santa Cruz, TX, USA), and the protein bands were photographed under a gel imager (Biorad, CA, USA).

The RIPA buffer (P0013 B, Beyotime) was used to extract proteins by lysing cells and tissues. The SDS-PAGE gel was used to separate the proteins. The proteins were transferred to the nitrocellulose membrane. 5% skimmed milk was used to block the membrane at 4°C overnight. The membranes were incubated with primary antibodies or secondary antibodies at room temperature (RT) for 90 min. The antibodies used were as follows: anti-β-actin (1 : 5000, 66009-1-Ig, proteintech), anti-PCNA (1 : 2000, 10205-1-AP, proteintech), anti-Ki67 (1 : 1000, 27309-1-AP, proteintech), anti-cleaved caspase 3 (1 : 1000, 9664S, CST), anti-caspase 9 (1 : 500, bs-20773R, Bioss Antibodies), anti-Bax (1 : 1000, ab32503, abcam), anti-Bcl-2 (1 : 1000, 12789-1-AP, proteintech), anti-E-cadherin (1 : 1000, 20874-1-AP, proteintech), anti-β-catenin (1 : 1000, bs-1165R, Bioss Antibodies), anti- N-cadherin (1 : 2000, 22018-1-AP, proteintech), anti-vimentin (1 : 2000, 10366-1-AP, proteintech), anti-Snail (1 : 1000, 13099-1-AP, proteintech), anti-Slug (1 : 1000, #9585, CST), HRP goat anti-mouse IgG (1 : 5000, SA00001-1, proteintech), and HRP goat anti-rabbit IgG (1 : 6000, SA00001-2, proteintech). Proteins were detected by Western Bright ECL kit (K-12045-D50, advansta). The expression of β-actin was applied as control.