Figure 7

CK-2-dpendent phosphorylation affects occludin solubility and dimerization. The distribution of wildtype (wt) occludin (Occ) and of the triple A (TripA) and E (TripE) mutated occludin constructs into TX-100-soluble and -insoluble fractions was analyzed by Western blotting (A) and quantified by chemoluminescence imaging (Occ-wt/Occ-TripA *p = 0,103; Occ-wt/Occ-TripE **p = 0,0018; n = 3) (B). C) Dimerization (cis-interaction) of occludin was analyzed by a fluorescence resonance energy transfer (FRET) assay in cell-cell contacts (see D) between two cotransfected HEK-293 cells. The constructs were N-terminally fused with cyan fluorescent protein (CFP, wt) and yellow fluorescent protein (YFP, mutants). Replacement of serine in human occludin at the position 408 by glutamate reduced the FRET efficiency to wt Occ (S408/wt, n = 40) compared to the wt/wt control (n = 58) similarly as the TripE mutant (TripE/wt, n = 44), whereas the TripA mutant was without any effect (TripA/wt, n = 64). **, p < 0.01, Mann–Whitney test. D) Fluorescence images of living HEK-293 cells cotransfected with the indicated CFP- and YFP-tagged Occ constructs used for the FRET measurements. Areas of FRET measurement at sites of cell-cell contacts are marked in red. The bar represents 10 μm.