Anti pan cadherin

Cells were fixed for 15 min in 4% paraformaldehyde at 37°C, washed three times in PBS and stored at 4°C prior to immunostaining. The antibodies were anti-N-cadherin (BD Biosciences, 610920, 1 : 250) or anti-Pan-cadherin (Sigma, C-1821, 1 : 100). F-actin staining was carried out with 647N-Phalloidin (Sigma, 65906, 1 : 300). Cells were washed twice in PBT (0.1% Tween20 in PBS), blocked for 30 min and then incubated for 2–3 h with primary antibodies in 2.5% goat serum, 1% DMSO in PBT. After washing eight times in PBT during 1 h, cells were incubated for 2 h in secondary antibody (donkey anti-mouse Cy5 conjugated, Jackson ImmunoResearch, 1 : 400) together with DAPI nuclear counterstain. The cells were then washed eight times in PBT and mounted in FluorSave. Immunostained cells were imaged using a Zeiss LSM710 confocal microscope and images processed using ImageJ. Fiji was used to plot the intensity profile of cadherin and F-actin staining in boundary assays. Western blotting was carried out with N-cadherin (1 : 1000) or Pan-cadherin (1 : 500) antibodies.

SCE cells were cultured on collagen-coated 8 well chamber slides at a density of 1 × 10⁴ cells per well in DMEM containing 10% FBS. Following overnight culture, when cells had reached confluence, K-115 at 1, 5, or 25 μmol/L, Y-27632 at 5 or 25 μmol/L, or fasudil at 5 or 25 μmol/L were added to culture media and cells were incubated for 30 min. PBS was used as a control vehicle. Cells were fixed with 4% paraformaldehyde in PBS for 15 min then washed with cytoskeletal buffer and serum buffer. Cells were permeabilized with 0.5% Triton X-100 in PBS for 12 min at room temperature. After washing in serum buffer, cells were blocked with serum buffer for at least 2 h at 4 °C. Cells were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-ZO-1 (1.25 μg/mL; Invitrogen, Carlsbad, CA), anti-β-catenin (1:1000 dilution; Sigma-Aldrich), and anti-pan-cadherin (1:100 dilution; Sigma-Aldrich). Cells were washed in serum buffer and then incubated with secondary antibody (Alexa Fluoro 488; Invitrogen) for 30 min at room temperature. After washing in PBS, cells were mounted with mounting medium contained DAPI and observed using a fluorescence microscope. The exposure to take images for ZO-1, β-catenin, pan-cadherin and DAPI were 0.5, 0.5, 0.8 and 0.05 sec, respectively.

For immunofluorescent analysis, kidneys were harvested and fixed in 4% formaldehyde overnight at 4°C, and then embedded in OCT (optimal cutting temperature compound) to obtain frozen-sections at 100 μm on Micron HM550 cryostat. The primary antibodies used were anti-Cox2 (100034-lea, Cayman), anti-Pax2a (ab229318, Abcam), anti-β-catenin (C7207, Sigma), anti-p-Ser9-GSK3 beta (ab107166, Abcam), anti-Pan-cadherin (C3678, Sigma), and anti-phospho-β-catenin (ser675) (D2F1) XP Rabbit mab (4176T, CST). The secondary antibodies used goat anti-mouse IgG H&L Alexa Fluor 647 (ab150115, Abcam), donkey anti-goat IgG Alexa Fluor 647 (ab150131, Abcam), goat anti-rabbit IgG (H+L) Alexa Fluor 633 (A11008, Invitrogen), and goat anti-rabbit IgG (H+L) Alexa Fluor 488 (A21070, Invitrogen), at 1:500. Images were taken using the Nikon A1 confocal microscope. Fluorescent intensities per unit area were measured using ImageJ.