Fig. 3. Expression of wild-type and mutant claudin-14 and other tight-junction proteins in MDCK cells. Stably transfected Tet-off MDCK cells were plated on filters and induced or not for 4 days; filters were subjected to electrophysiological measurements and then protein was extracted by incubating filters directly into SDS sample buffer. Samples were electrophoresed and immunoblotted. Lanes: (+), with doxycycline (noninduced); (–), without doxycycline (induced). No endogenous claudin-14 could be detected in MDCK cells using either anti-human- or anti-mouse-claudin-14 antibodies. Transgene expression is tightly regulated by doxycycline. Expression of claudin-2, ZO-1, cadherin and tubulin were unaffected by claudin-14 expression, whereas the levels of both claudin-4 and occludin were downregulated by transgene induction. This immunoblot represents results from a single clone of the wild type and each mutant, but induction of transgenes were verified in all clones used in physiological experiments (three to six for the wild type and each mutant).