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Fig. 4. (A) TER of MDCK cells expressing wild-type and mutant claudin-14. Tet-off MDCK cells were cultured on filters and induced, or not, to express wild-type and palmitoylation-deficient claudin-14. Light gray bars indicate uninduced MDCK cells; dark gray bars indicate induced MDCK cells. Values are means ± s.e.m. of three to six separately derived clonal cell lines for wild-type claudin-14 and each mutant. Expression of wild-type claudin-14 in MDCK cells results in a fivefold increase in TER. Mutation of either of the palmitoylation sites singly or in combination decreases the ability of claudin-14 to raise the TER. *, P<0.05 compared with wild-type as determined by ANOVA followed by Dunnett's test. (B) Effects on TER of graded induction of wild-type claudin-14 (open circles) and 4S mutant protein (closed circles). Tet-off MDCK cells stably expressing the transgenes of claudin 14 and mutants were variably induced to express transgenes by using different doses of doxycycline. Because the same antibody was used to detect both wild-type and mutant claudin-14, protein levels could be directly compared after quantification using the Li-Cor Odyssey detection system (x-axis marked with arbitrary units). Although the levels of the 4S mutant could be induced to more than four-times-higher levels than in wild-type claudin-14, there was no significant increase in TER in MDCK cells expressing the mutant protein.
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