Mutation (_mut) in the extracellular loop (ECL) 2 leads to decreased cis- and trans-interaction of claudins (Cldns).

A,B:trans-Interactions assessed by transepithelial electrical resistance (TER) and permeability in filter-grown Madin-Darby canine kidney cells II expressing murine Cldn5_{wild type} (wt), Cldn5_F147A, Cldn5_Q156E or untransfected (-). A: TER-values, n≥8. B: Permeability coefficient for lucifer yellow across the monolayer, n = 9. C: Homophilic cis-interaction between yellow fluorescent protein (YFP)/mTurquoise2 pairs of murine Cldn5_wt or Cldn5_mut was measured as fluorescence resonance energy transfer (FRET)-efficiency along the plasma membrane between transiently transfected HEK-293 cells. CRFR: Corticotropin-releasing factor receptor 1 (negative control). Values were normalized to Cldn5_wt, n>25. D: Mobility of YFP-Cldn5 in the membrane measured by fluorescence recovery after photobleaching (FRAP). MF: mobile fraction, calculated from fitted curve. n>26. Mean±SEM, *p<0.05, **p<0.01, ***p<0.001, ns: not significant. Kruskal-Wallis test with Dunn’s post hoc test. Differences shown compared to Cldn5_wt transfected (*) or untransfected cells (#).