Figure 2.
Effect of ADAM10 inhibitor on VE-cadherin cleavage induced by FXIa. HUVECs were incubated with FXIa for 6 hours in the presence or absence of the serine protease inhibitor, PPACK (100 μM); the thrombin inhibitor, hirudin (25 μg/mL); or the ADAM10 inhibitor, GI254023X (10 μM). (A) Cells were lysed and analyzed by western blotting using an anti–C-terminal VE-cadherin antibody. Results are representative of 3 experiments. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3). (B) Cell media were analyzed by using a sVE cadherin enzyme-linked immunosorbent assay. Data are mean ± SEM (n = 3). Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. (C) Cell media were also analyzed by western blot using an anti–N-terminal VE-cadherin antibody. Results representative of 3 experiments. (D) HUVECs were grown on gelatin-coated glass coverslips and incubated with FXIa (30 nM) for 6 hours in the absence or presence of GI254023X (10 μM). Cells were fixed and stained for VE-cadherin (green), actin (red), and nuclei (blue). Bar, 50 μm. (E) HUVECs were grown to confluence on gelatin-coated Transwell filters and incubated with FXIa (30 nM) in the absence or presence of GI254023X (10 μM). Permeability of Evans Blue-BSA was measured after 60 minutes of incubation. Data are mean ± SEM (n = 4). Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. (F) HUVECs were incubated with FXIa (30 nM) for 6 hours in the absence or presence of PPACK (100 μM). Cell surface was biotinylated and cell lysates were precipitated with NeutrAvidin agarose beads and probed with an anti-ADAM10, anti–N-terminal VE-cadherin, or anti-PECAM1 antibodies. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3).

Effect of ADAM10 inhibitor on VE-cadherin cleavage induced by FXIa. HUVECs were incubated with FXIa for 6 hours in the presence or absence of the serine protease inhibitor, PPACK (100 μM); the thrombin inhibitor, hirudin (25 μg/mL); or the ADAM10 inhibitor, GI254023X (10 μM). (A) Cells were lysed and analyzed by western blotting using an anti–C-terminal VE-cadherin antibody. Results are representative of 3 experiments. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3). (B) Cell media were analyzed by using a sVE cadherin enzyme-linked immunosorbent assay. Data are mean ± SEM (n = 3). Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. (C) Cell media were also analyzed by western blot using an anti–N-terminal VE-cadherin antibody. Results representative of 3 experiments. (D) HUVECs were grown on gelatin-coated glass coverslips and incubated with FXIa (30 nM) for 6 hours in the absence or presence of GI254023X (10 μM). Cells were fixed and stained for VE-cadherin (green), actin (red), and nuclei (blue). Bar, 50 μm. (E) HUVECs were grown to confluence on gelatin-coated Transwell filters and incubated with FXIa (30 nM) in the absence or presence of GI254023X (10 μM). Permeability of Evans Blue-BSA was measured after 60 minutes of incubation. Data are mean ± SEM (n = 4). Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. (F) HUVECs were incubated with FXIa (30 nM) for 6 hours in the absence or presence of PPACK (100 μM). Cell surface was biotinylated and cell lysates were precipitated with NeutrAvidin agarose beads and probed with an anti-ADAM10, anti–N-terminal VE-cadherin, or anti-PECAM1 antibodies. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3).

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