Anti e cadherin

Cells were seeded and incubated overnight, and then divided into 2 groups and switched to media with or without 5 ng/mL TGF-β. After incubation for 24 h, nanaomycin K (5 µg/mL or 50 µg/mL) or 0.05% DMSO was added to the cultures. After incubation for an additional 48 h, cells were washed and lysed in 8 M urea buffer. Each sample was added into sample buffer (Nacalai Tesque, Kyoto, Japan) and heated at 95 °C for 5 min. The samples were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with Blocking One or Blocking One-P (Nacalai Tesque) followed by washing, the membranes were incubated overnight at room temperature with anti-E-cadherin (Biolegend, San Diego, CA), anti-N-cadherin (Biolegend), anti-vimentin (Biolegend), anti-phospho-p38 MAPK (Thr180/Tyr182) (Cell Signaling Technology: CST, Danvers, MA), anti-phospho-SAPK/JNK (Thr183/Tyr185) (CST), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (CST), anti-Snail (CST), anti-Slug (CST) or anti-β-actin (Santa Cruz Biotechnology, Dallas, TX), respectively. After another washing, membranes were incubated for 1 h with HRP-conjugated secondary antibodies. Antibody binding to proteins was detected by enhanced chemiluminescence.

Whole mouse kidneys and intestine were perfused with cold saline, rinsed, and resected. Single-cell suspensions were generated in Multi-Tissue Dissociation Kit-2 on a gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) using the 37MTDK-2 setting. Suspensions were then filtered through 40-μm nylon strainers, rinsed in saline, and fractionated across a 9-ml 25–40–60% Percoll (GE Healthcare) density gradient. The lower layer corresponded to “parenchymal” resident cells and the upper layer to immune cell populations and mesangial cells. Separate fractions were then incubated in Fc block at 1 μg/10⁶ cells for 5 min at room temperature, followed by primary antibody labeling with anti-CD45 (clone 30-F11; BioLegend), anti-E-cadherin (Cat. no. 147303; BioLegend), anti-CD3 (clone 145-2C11; Invitrogen), anti-IgM (clone EB121-15F9; Invitrogen), anti-Ly6G (clone HK1.4; BioLegend), and anti-F4/80 (clone BM8; BioLegend). Flow cytometry was performed using hierarchical gating on an FACSARIA III (BD Biosciences), and RNA was isolated from sorted epithelial cells by Solution D.

Flow cytometry was used to determine the surface expression of ADAM10, EGFR and E-cadherin on 16HBE14o- and S9- cells. For flow cytometric analysis, cells were trypsinized and 1 x 10⁶ cells were stained with PE-conjugated anti-ADAM10 (BioLegend), anti-E-cadherin (BioLegend), anti-EGFR (BioLegend) or appropriate isotype control (IgG1k, eBioscience) antibodies in 100 μl medium for 30 min at 4°C in the dark. Cells were washed twice with FACS buffer (DPBS, 1% (v/v) FBS, 3.8 mM sodium azide) and resuspended therein. Stained cells were analyzed on an Attune Acoustic Focusing Cytometer (Life Technologies). Ten thousand events were gated and analyzed with Attune software V2.1.0 or FlowJo V10.07 (Tree Star).