Characterization of ZO-1 constructs and claudin/JAM-A/occludin KO and claudin/JAM-A/CAR KO cells. (A) ZO1-GFP was able to restore the continuous apical junction localization of occludin (arrows) upon expression in ZO-1/ZO-2 double KO cells. (B) HA-ZO1-Flag was able to rescue the continuous apical junction localization of occludin (arrows) upon expression in ZO-1/ZO-2 double KO cells. (C) Western blotting of occludin, CAR, and α-tubulin in MDCK II, claudin/JAM-A KO, claudin/JAM-A/occludin KO, and claudin/JAM-A/CAR KO (clones 1 and 2) cells. Occludin expression was specifically lost in claudin/JAM-A/occludin KO cells, while CAR expression was specifically lost in claudin/JAM-A/CAR KO cells. Scale bar: 10 μm. Source data are available for this figure: SourceData FS5.