Anti n cadherin

For detecting exosome-associated surface proteins, on-bead flow cytometry was performed following exosome capture on streptavidin magnetic beads coated with biotin- labeled anti-CD63 mAbs. The detailed protocol was previously reported [47 (link)]. The following labeled detection Abs were used for staining: anti-E-cadherin (Biolegend, # 324104), anti-N-cadherin (Biolegend # 350808) or and anti-TGF-β (R&D, # FAB2463P). Immediately following 1h staining with Abs at RT and washing with buffer, flow cytometry was performed using a Gallios instrument. Samples were run for 2min and 10,000 events were acquired. For each detection Ab, corresponding isotype controls were included.

Cells were kept in culture for 72 hours. Cells collected for flow cytometry were washed with 1x PBS, trypsinised, and resuspended in PBS-2% BSA-0.05% Tween® 20. One million (1 x 10⁶) cells were used for each labelling. The primary antibodies used were: anti-CD49f-PE (BD, Pharmigen, Franklin Lakes, NJ, USA); anti-EpCAM, anti-MUC1, anti-EGFR, anti-N-cadherin, anti-E-cadherin, anti-CD66a/c/e (Biolegend, San Diego, CA, USA); anti-EpHB4 and anti-claudin-3 (R&D Systems, Inc., MN, USA); anti-vimentin, anti-claudin-4, anti-ALDH1, anti-FGFR; secondary antibodies were Alexa488 and Alexa647 (Abcam, Cambridge, UK). Unlabelled cells and with only secondary antibody labelling were included in each independent assay and considered autofluorescent. Dead cells and debris were excluded from the analysis. Cells were analyzed in a FACs-Aria flow cytometer (BD Bioscience).

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.