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. 2011 Apr 6;286(22):19641–19651. doi: 10.1074/jbc.M110.186239

FIGURE 2.

FIGURE 2.

Amentoflavone inhibits VEGFR phosphorylation and HUVEC migration. A, representative pictures of Western blot analysis of VEGFR-1 phosphorylation (P-VEGFR-1) induced by 20 ng/ml of PlGF-1 or 50 ng/ml VEGF-A on cells overexpressing VEGFR-1 (293-Flt-1). AF was used at a concentration ranging between 2 and 25 μm. As control of inhibition neutralizing antibody anti-PlGF (α-PlGF) or anti-VEGF-A (α-VEGF-A) were used at 3.3 nm. Anti-VEGFR-1 antibody was used for normalization. B, Western blot analysis of VEGFR-2 phosphorylation (P-VEGFR-2) performed in the same condition as reported in A but on HUVECs. Anti-VEGFR-2 antibody was used for normalization. Histograms represent densitometry analysis of three independent experiments, and data are represented as the mean ± S.E. ADU, arbitrary densitometric unit. *, p < 0.0005 versus PlGF-1 or VEGF-A; §, p < 0.001 versus VEGF-A; #, p < 0.005 versus PlGF-1 or VEGF-A. C, cell migration induced by VEGF-A or PlGF-1 was significantly inhibited by AF used at 10 and 50 μm. Conversely, AF failed to inhibit FGF-2-stimulated cell migration D, 0% or 5% FCS were used as negative and positive control of migratory stimulus. *, p < 0.0001 versus VEGF-A, PlGF-1, and FCS (5%). Each point was done in quadruplicate, and the experiments were performed twice. Data are represented as the mean ± S.E.