Reduction of VEGF-A–induced p-Y1175 VEGFR-2 by a Syk inhibitor. (A) HDMECs were serum-starved overnight and washed with PBS (supplemental Figure 5A). Cells were incubated with 0.5% FBS in PBS with or without 4.7μM BAY 61-3606 (Syk inhibitor), as described in the endothelial kinase assay section in “Methods.” Cells were lysed in 1% Triton X-100, and an equal amount of protein was used for immunoblotting using Abs against p-Y323 Syk, or p-1054 VEGFR2 or p-Y1175 VEGFR-2. Inhibition of Syk did not affect the autophosphorylation of VEGFR-2 at Y1054, but it decreased phosphorylation of Y1175 in response to VEGF-A. (B) The experiment above was repeated using HUVECs that were infected with either control vector or full-length CD36 vector (supplemental Figure 4B). Cells were treated as described in panel A and phosphorylation of Syk and VEGFR-2 was detected. The results showed that Syk inhibitor reduced phosphorylation of VEGFR-2 at Y1175, but did not affect the level of p-Y1054 in cells with overexpression of CD36. The level of both p-Y1175 and p-Y1054 remained unchanged in control cells. These experiments were done independently 3 times.