Figure 5.
Role of FXIa on Dab1 and VEGFR2 activation. (A) HUVECs were incubated with FXIa (30 nM) in the absence or presence of (A) the Src inhibitor, PP2 (10 μM), or the low-density lipoprotein receptor antagonist, RAP (50 ng/mL). Cells were lysed and immunoblotted with antibodies for Dab1 Y2020, PLCγ1 Y783, or tubulin. Results are representative of 3 experiments. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. (B) HUVECs were incubated with FXIa (30 nM) in the absence or presence of the VEGFR2 inhibitors, BFH772 (1 μM) or SU 1498 (5 μM). Cells were lysed and immunoblotted with antibodies for Dab1 Y2020, Src Y416, PLCγ1 Y783, or tubulin, or lysates were separated by Phos-tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted for VEGFR2 phosphorylation (n = 3). Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3). (C) HUVECs were incubated with vehicle or FXIa for 6 hours in the absence or presence of the VEGFR2 kinase activity inhibitor, SU1498 (5 μM), or the blocking anti-VEGF antibody, ramucirumab (10 μg/mL). 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). (D) HUVECs were grown to confluence on gelatin-coated Transwell filters and incubated with FXIa (30 nM), for 6 hours in the absence or presence of PP2 (10 μM) or SU1498 (5 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. (E) HUVECs were incubated with FXIa (30 nM) for 6 hours in the absence or presence of the ADAM10 inhibitor, GI254023X (5 μM), or the Src inhibitor, PP2. Cell media were collected and immunoblotted with antibodies for N-terminal VE-cadherin or N-terminal VEGFR2. Results are representative of 3 experiments.

Role of FXIa on Dab1 and VEGFR2 activation. (A) HUVECs were incubated with FXIa (30 nM) in the absence or presence of (A) the Src inhibitor, PP2 (10 μM), or the low-density lipoprotein receptor antagonist, RAP (50 ng/mL). Cells were lysed and immunoblotted with antibodies for Dab1 Y2020, PLCγ1 Y783, or tubulin. Results are representative of 3 experiments. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. (B) HUVECs were incubated with FXIa (30 nM) in the absence or presence of the VEGFR2 inhibitors, BFH772 (1 μM) or SU 1498 (5 μM). Cells were lysed and immunoblotted with antibodies for Dab1 Y2020, Src Y416, PLCγ1 Y783, or tubulin, or lysates were separated by Phos-tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted for VEGFR2 phosphorylation (n = 3). Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3). (C) HUVECs were incubated with vehicle or FXIa for 6 hours in the absence or presence of the VEGFR2 kinase activity inhibitor, SU1498 (5 μM), or the blocking anti-VEGF antibody, ramucirumab (10 μg/mL). 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). (D) HUVECs were grown to confluence on gelatin-coated Transwell filters and incubated with FXIa (30 nM), for 6 hours in the absence or presence of PP2 (10 μM) or SU1498 (5 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. (E) HUVECs were incubated with FXIa (30 nM) for 6 hours in the absence or presence of the ADAM10 inhibitor, GI254023X (5 μM), or the Src inhibitor, PP2. Cell media were collected and immunoblotted with antibodies for N-terminal VE-cadherin or N-terminal VEGFR2. Results are representative of 3 experiments.

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