Fig. 2. Behavior of AJ and TJ constituents in Snail-overexpressed epithelial cells. (A) Immunoblotting of Eph4 cells, two independent Eph4-mSnail clones and NIH/3T3 cells with antibodies specific for AJ and TJ components. In Eph4-mSnail cells, E-cadherin, claudin-3 and occludin became undetectable at the protein level. By contrast, the expression levels of p120 and ZO-1 (E-cadherin- and claudin/occludin-binding undercoat protein, respectively) did not appear to be altered significantly by the Snail overexpression, although p120 showed significant mobility shifts for unknown reasons. The expression pattern of AJ and TJ components in Eph4-mSnail cells is very similar to that in NIH/3T3 fibroblasts. Cytokeratin-18 (CK18) is an epithelial marker. (B) Immunofluorescence microscopy of Eph4 and Eph4-mSnail cells with antibodies specific for AJ and TJ components. In Eph4-mSnail cells, E-cadherin as well as claudin-3/occludin became undetectable not only in the cell-cell contact regions but also in the cytoplasm. By contrast, Snail appeared to translocate ZO-1 and p120 from the junctional regions to the cytoplasm. Bars, 30 µm.