FIGURE 1.

TSP1 and a CD47-binding peptide inhibit VEGFR2 signaling without inhibiting VEGF binding. A, TSP1 suppresses VEGFR2 phosphorylation. A representative Western blot is shown using anti-Tyr¹¹⁷⁵ VEGFR2 and anti-VEGFR2 antibodies with lysates from untreated BAEC (lane 1) and BAEC stimulated with VEGF (30 ng/ml) for 5 min (lane 2), treated with TSP1 (2 nm) alone (lane 3), and pretreated with TSP1 (2 nm) followed by VEGF (30 ng/ml) for 5 min (lane 4). B, quantification of VEGFR2 phosphorylation in three independent experiments normalized to loading controls. p values were as follows: VEGF versus untreated (UT), 0.00007 (*); and VEGF versus TSP1 + VEGF, 0.0001 (**). C, inhibition of VEGFR2 phosphorylation by TSP1 in HDMVEC (quantification of two independent experiments). p values were as follows: untreated versus VEGF, 0.09 (*); VEGF versus TSP1 + VEGF, 0.07 (**); VEGF versus TSP1, 0.08; and untreated versus TSP1, 0.7. D, iodinated VEGF binding to BAEC was assessed in the presence of the indicated concentrations of TSP1 or in the presence of a 200-fold excess of unlabeled VEGF. E, TSP1 and 7N3 inhibition of VEGF binding to BAEC. F, inhibition of VEGFR2 phosphorylation by peptide 7N3. Lane 1, untreated; lane 2, 7N3 (1 μm) alone; lane 3, 7N3 (1 μm) and VEGF (30 ng/ml); lane 4, VEGF (30 ng/ml)-stimulated. G, quantification of three independent experiments. p values were as follows: untreated versus VEGF; 0.007 (*); and VEGF versus 7N3 + VEGF, 0.012 (**).