Gtx100443

Immunofluorescence was performed as described previously (13 (link)). The following primary antibodies were used: anti-NFκB (GTX102090, GeneTex), anti-β-catenin (E247, Epitomics #1247-s), anti-E-cadherin (GTX100443, GeneTex), and anti-N-cadherin (TA326835, OriGene). Goat anti-rabbit secondary antibody was coupled to AlexaFluor 555 (Invitrogen). Images were obtained with DS-Ri1 Nikon camera and Eclipse80i Nikon microscope and quantifications were performed using NIS-Elements BR 4.20.00 software (Nikon).

Total protein was extracted from the renal cortex of rats and quantified with the BCA assay (MultiSciences Biotech Co., Ltd., Hangzhou, China). Then the protein was diluted with 5× loading buffer and denatured at 100°C for 5 minutes. Afterwards, the protein samples were electrophorised in SDS polyacrylamide gel and then transferred onto PVDF membranes (Millipore Corporation, Bedford, MA, USA). The nonspecific background bindings were blocked with 5% nonfat milk for an hour. Then the membranes were incubated in primary antibodies at 4°C overnight and secondary antibodies for 1 hour. Optical density of the bands was scanned by Odyssey infrared fluorescence imaging system (LI-COR, USA) and quantified using ImageJ (National Institutes of Health, USA). The primary antibodies used are as follows: Wnt4 (sc-376279, Santa Cruz Biotechnology, Inc., USA, 1: 500), TCF4 (sc-166699, Santa Cruz Biotechnology, Inc., USA, 1: 500), GSK3β (ab93926, Abcam, Inc., UK, 1: 1000), p-GSK3β (ab131097, Abcam, Inc., UK, 1: 1000), E-Cadherin (GTX100443, GeneTex Inc., CA, USA, 1: 1000), ɑ-SMA (ET1607-43, Hangzhou HuaAn Biotechnology Co., Ltd., Hangzhou, China, 1: 5000), and Vimentin (ab92547, Abcam, Inc., UK, 1: 5000).