Research Article
Formyl Peptide Receptor 1 Modulates Endothelial Cell Functions by NADPH Oxidase-Dependent VEGFR2 Transactivation
Figure 4
FPR1-mediated Flk-1/KDR transactivation triggers the PI3K-Akt pathway. (a, c) Cell lysates were purified from serum-starved ECV304 cells exposed to 0.1 μM N-fMLP for the indicated times. (b, e) ECV304 cells were serum-deprived for 24 hours before the stimulation for 5 minutes with N-fMLP in the absence or presence of PTX, apocynin, (d) wortmannin, or LY294002. (f) ECV304 cells were serum-deprived for 24 hours, incubated for 12 hours with 5 nM siRNA against FPR1 (FPR1 siRNA) or (g) against p22phox (p22phox siRNA) or negative control siRNA (NC siRNA), in DMEM containing 10% FBS in the presence of 20 μl of HiPerfect, and stimulated with 0.1 μM N-fMLP for 5 minutes. Fifty micrograms of whole lysates was resolved on 10% SDS-PAGE and immunoblotted with (a, b) an α-pPI3K (p85) or (c–g) an α-pAkt (S473) antibody. An α-tubulin antibody served as a control for protein loading. All the experiments are representative of at least three independent experiments. compared with unstimulated cells. compared with unstimulated cells.
| (a) |
| (b) |
| (c) |
| (d) |
| (e) |
| (f) |
| (g) |