Fig. 3. CNF-1 alters morphological localization of candidate TJ proteins. T84 monolayers were exposed to CNF-1 or vehicle alone for 24 hours, and TJ proteins occludin, ZO-1 and JAM were localized by immunofluorescence and confocal microscopy. In control monolayers (a), occludin localized sharply in the apical region of the lateral membrane, visualized en face as a ring pattern. Monolayers treated apically for 24 hours with CNF-1 (b) were identical. Monolayers exposed basolaterally to CNF-1 for 24 hours (c) displayed dramatic redistribution of occludin away from the lateral TJ membrane (arrow). ZO-1 staining in control monolayers (d) resembled that of occludin, outlining TJs just below the apical plane. Apical exposure to CNF-1 for 24 hours did not affect ZO-1 immunolocalization (e); however, discontinuities in ZO-1 ring structures were evident in monolayers treated basolaterally with CNF-1 for the same time period (f). JAM in control monolayers (g) was enriched in the TJ plane, and unchanged by apical treatment with CNF-1 toxin for 24 hours (h). However, basolateral exposure to CNF-1 for the same time period (i) somewhat disrupted JAM distribution, with diffusion away from the TJ membrane into the cytoplasm. Levels of occludin (j), ZO-1 (k) or JAM (1) proteins were minimally different in lysates prepared from control (lane 1) versus CNF-treated (lane 2) epithelial monolayers. Bar10 µm.