Mutation (<sub>mut</sub>) in the extracellular loop (ECL) 2 leads to decreased <i>cis</i>- and <i>trans</i>-interaction of claudins (Cldns).

<p><b>A,B:</b><i>trans</i>-Interactions assessed by transepithelial electrical resistance (TER) and permeability in filter-grown Madin-Darby canine kidney cells II expressing murine Cldn5<sub>wild type</sub> (wt), Cldn5<sub>F147A</sub>, Cldn5<sub>Q156E</sub> or untransfected (-). <b>A</b>: TER-values, n≥8. <b>B</b>: Permeability coefficient for lucifer yellow across the monolayer, n = 9. <b>C</b>: Homophilic <i>cis</i>-interaction between yellow fluorescent protein (YFP)/mTurquoise2 pairs of murine Cldn5<sub>wt</sub> or Cldn5<sub>mut</sub> 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<sub>wt</sub>, n>25. <b>D:</b> 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<sub>wt</sub> transfected (*) or untransfected cells (#).</p>