Formyl Peptide Receptor 1 Modulates Endothelial Cell Functions by NADPH Oxidase-Dependent VEGFR2 Transactivation

Figure 2

FPR1 activation promotes Flk-1/KDR transphosphorylation. (a) Whole lysates (900 μg) purified from serum-starved ECV304 cells stimulated with 0.1 μM N-fMLP for the indicated times were immunoprecipitated with an α-Flk-1 antibody, and Flk-1 tyrosine phosphorylation was detected with an α-pY antibody. An α-Flk1 antibody served as a control for protein loading. (b, c, d) Fifty micrograms of whole lysates, purified from ECV304 cells, was resolved on 10% SDS-PAGE. The cells were (b) stimulated with N-fMLP for the indicated times, (c) stimulated with N-fMLP for 5 minutes in the presence or absence of PTX, or (d) incubated for 12 hours with 5 nM siRNA against FPR1 (FPR1 siRNA) or negative control siRNA (NC siRNA) in DMEM containing 10% FBS in the presence of 20 μl of HiPerfect. The filters were immunoblotted with α-pFlk-1 (Y951), α-pFlk-1 (Y996), α-pFlk-1 (Y1175), or α-pFlk-1 (Y1214) antibodies. An α-Flk-1 antibody served as a control for protein loading. compared with unstimulated cells. compared with unstimulated cells. compared with unstimulated cells.

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