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Fig. S4 Overexpression of EpCAM in MDCK cells causes reduced levels of tight junction proteins. MDCK cells were transfected with expression vectors driving either GFP (A–C, G–I, M–O) or zebrafish EpCAM-GFP expression (D–F, J–L, P–R), plated on cover slips after FACS sorting one day after transfection, and stained for cell-cell junctional markers 48 hours after plating. Processed confocal images are shown as merges of entire Z-stacks. Transfectants were detected by GFP (B, H, N) or EpCAM-GFP expression (E, K, Q). Protein localization was analyzed with antibodies against Tjp1/ZO-1 (C, F), Occludin (Occ) (I, L), or Desmoplakin 1 and 2 (DPI/II) (O, R); panels (A,D,G,J,M,P) show overlays with GFP fluorescence. DAPI-stained nuclei are shown in blue (A,D,G,J,M,P); scale bar, 10 μm. Fluorescent labeling of key components of tight junctions reveals a reduction in membrane localization of Tjp1 in EpCAM- (F) versus GFP-transfected (C) cells. Similarly, Occludin staining is reduced in EpCAM expressing MDCK cells (I, L). However, proteins localizing to adherens junctions or desmosomes are not altered in expression or cellular distribution, as shown for DPI/II (O, R). This indicates that EpCAM expression in MDCK cells leads to a modification in apical junction complex assembly, resulting in a reduction of key tight junction proteins at the plasma membrane. (S) Immunoblot of lysats from FACS-sorted MDCK cells transfected with EpCAM-GFP (right lane) or, as control, GFP (left lane). EpCAM-GFP and GFP are expressed at comparable levels (upper panel). Expression of EpCAM-GFP leads to a significant in Tjp1/ZO1 protein levels (middle panel; arrow indicate full-length Tjp1/ZO1 protein). Gapdh was used as loading control (lower panel).