Effect of ANXA2 on VEC phosphorylation. (A) After stimulation with rVEGF (0–3 h) in the presence or absence of the Src inhibitor PP2, or control PP3, extracts of confluent Anxa2^+/+ and Anxa2^−/− CMECs were immunoblotted with anti–pY731-VEC, -pY416-Src-, -VEC, -Src, -ANXA2, and -actin. (B and C) Permeability to FITC–dextran and TEER of CMECs treated with or without recombinant VEGF in the presence or absence of PP2 or PP3 (n = 6 samples per group, ANOVA). (D) Lysates from VEGF-treated Anxa2^+/+ and Anxa2^−/− CMECs were immunoblotted with anti–pY731-VEC (pY731-73.1, courtesy of D. Vestweber; Wessel et al., 2014), -VEC, -ANXA2, and –pan-cadherin (Pan-cad). (E) Quantification of blots described in D (combined data from 11 experiments, ANOVA). (F) Lysates were blotted with two anti–pY685-VEC antibodies (Ab#1: ab119785; Abcam; and Ab#2: PhoCad3.3, courtesy of D. Vestweber; Wessel et al., 2014), as well as anti-VEC and anti-ANXA2. (G) Lysates were blotted with anti-pY992-Tie2, -Tie2, -ANXA2, and –pan-cadherin. Data in A–C, F, and G are representative of three experiments, and data in D and E are representative of 11 experiments. Black and white bars indicate Anxa2^+/+ and Anxa2^−/−, respectively. Data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.