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Fig. 9. (A) Localization of wild-type and the 4S mutant of claudin-14 at 4 days, 8 days and 12 days in culture. Filters used for determination of TER (below) were processed for immunofluorescent analysis. As shown in Fig. 5, wild-type claudin-14 localizes to cell borders and small intracellular puncta after 4 days of induction, and this localization is unaltered after 8 days or 12 days in culture. The 4S mutant of claudin-14 is found in large intracellular aggregates at day 4 but, at days 8 and 12, this same claudin-14 mutant is predominantly localized to cell borders, similar to the wild-type distribution. (B) TER of MDCK cells expressing wild-type and the 4S mutant of claudin-14 after extended periods of time in culture. Tet-off MDCK cells were cultured on filters as described previously and induced or not to express wild-type (uninduced, black bars; induced, dark gray bars) and the 4S mutant of claudin-14 (uninduced, white bars, induced, light gray bars); values are means ± range for duplicate wells. TER was determined in replicate filters at 4 days, 8 days and 12 days. At 4 days, expression of wild-type claudin-14 resulted in increased TER similar to that shown in Fig. 4, whereas expression of this 4S mutant resulted in decreased TER relative to uninduced control. After 8 days and 12 days of induction, TER was increased three to five times in both the wild-type and 4S-expressing cells.
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