Phosphorylation of T400/T404/S408 differentially affects binding to ZO-1 and ZO-2. A) The indicated occludin (Occ) constructs were transiently cotransfected into HEK-293 cells. The phosphomimetic Occ S408E shows reduced cis-interaction along the cell membrane with ZO-1 protein (S408/ZO-1, n = 12) compared to Occ wt with ZO-1 (wt/ZO-1, n = 26), p < 0.05 (*), Mann–Whitney test, one-tailed. The Occ triple E mutant (TripE/ZO-1, n = 30) as well as the triple A mutant (TripA/ZO-1; n = 34) were without effect. B) In contrast to ZO-1, phosphomimetic triple E and S408E occludin constructs exhibited reduced cis-interaction with the ZO-2 protein (tripleE/ZO-2, n = 37; S408/wt, n = 42) compared to Occ wt (wt/ZO-2, n = 35), p < 0.011 (***), Mann–Whitney test, one-tailed. The Occ mutant triple A (TripA/ZO-2; n = 32) was without significant effect. Cis-interaction was measured as fluorescence resonance energy transfer (FRET) efficiency using a FRET assay at cell-cell contacts between two cotransfected cells. The Occ constructs were N-terminal fusions with yellow fluorescent protein (YFP) and that of ZO-1 and ZO-2 were C-terminally fused with cyan fluorescent protein (CFP).