Fig. 2: CD31 signals prevent VE-cadherin and β-catenin phosphorylation in response to MHC stimulation via ITIM 686-thyrosine phosphorylation.

a phosphorylation of β-catenin (Y654) by MHC- or ICAM-1-stimulated (30 min) WT or cd31^−/− EC was analyzed by western blotting. The bar graph shows relative protein expression ± SEM. (N = 3 independent experiments). One-way Anova with Tuckey post-hoc test. cd31^−/− MHC vs all ***p = 0.0002. b–e phosphorylation of β-catenin (Y654, b) or VE-cadherin (Y685, d) by MHC-stimulated (30 min) WT or CD31^−/− EC was analyzed by widefield fluorescence microscopy. The bar graphs c, e show the mean fluorescence intensity/per cell of the indicated marker measured in three independent experiments by ImageJ software. Scale bar, 20 μm. Magnification ×20. Data are mean ± SD. One-way Anova with Tuckey post-hoc test. c cd31^−/− MHC vs all ****p < 0.0001; e cd31^−/− MHC vs all ****p < 0.0001. f, g CD31 gene constructs with mutations leading to the loss-of-function amino acid substitutions Y663F and Y686F in the ITIMs were generated and expressed by lentiviral transduction into cd31^−/− (KO) ECs (KO^CD31Y686F, KO^CD31Y663F). As a control, CD31 KO ECs were transduced with a wild-type CD31 gene construct (KO^CD31WT) or an empty plasmid (KO^pklo.1). ECs (6 × 10⁴/well) previously treated with 300 U/ml IFN-γ for 48 h (to enhance MHC molecule and ICAM-1 expression) were seeded onto 0.2 μm-pore transwells and stimulated with 5 μg/ml anti-mouse H-2Ld/H-2Db, or relevant isotype control followed by a secondary cross-linking Ab. TEER was measured as described in the Methods section. n = 3 biologically independent samples, N = 2 independent experiments. Data are mean ± SD. One-way Anova with Tuckey post-hoc test. f KO ^CD31WT vs all ***p  = 0.0002; g KO ^CD31WT vs all ***p = 0.0002, KO^Y663 vs. KO ^pklo.1 **p = 0.0024.