Characterization of claudin-2 knockout clones expressing exogenous claudin-2.

(A) Immunoblots of claudin-2 and E-cadherin in control MDCK II cells and claudin-2 knockout clones expressing exogenous claudin-2. Claudin-2 cDNA was transfected into claudin-2 knockout clone 1 (KO 1), and clones expressing FLAG tagged claudin-2 (clones F1–5) and tagless claudin-2 (clone Tl) were established. The signal intensity of claudin-2 bands was quantified, and relative signal intensity was calculated as the ratio of the signal intensity in each clone to that in control cells. N = 4 for each experiment. (B) Immunofluorescence analysis of claudin-2 and FLAG in co-culture of control MDCK II cells and F1–5 clones. Claudin-2 staining was barely detectable at TJs in F1–3 clones, whereas claudin-2 staining in the F4 and 5 clones was clearly detected at TJs but signals were weaker compared with control cells. Scale bar = 10 μm.