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. Author manuscript; available in PMC: 2016 Jan 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2014 Nov 6;35(1):111–120. doi: 10.1161/ATVBAHA.114.304554

Figure 2.

Figure 2

Inhibition of VEGFR-2 phosphorylation by PAI-1 depends of VN and endocytic receptor binding. (A) HUVECs were transfected with human PAI-1 siRNA expression vector or control (Con-siRNA) plasmid. Western blot analysis of PAI-1 and β-actin expression in transfected HUVECs is shown. (B) PAI-1-silenced HUVECs were incubated with recombinant PAI-1 (WT, AKE, E, R, or AK mutant) or vehicle control (-) and stimulated with VEGF (+, 50 ng/mL) or vehicle control (-), as shown. Cell lysates were prepared and phosphorylated (p) and total VEGFR-2 were assessed by Western blotting. (C) Pre-incubation of HUVECs with RAP (0.2 μM) blocks PAI-1-AK's capacity to inhibit VEGF-induced VEGFR-2 phosphorylation. All graphs correspond to blots above them and represent densitometric analyses of 3 independent experiments. *P<0.05 vs. negative control (1st bar).