Research Article
Formyl Peptide Receptor 1 Modulates Endothelial Cell Functions by NADPH Oxidase-Dependent VEGFR2 Transactivation
Figure 1
ECV304 cells express Flk-1/KDR, FPR1, and NADPH oxidase. Total RNA was purified from ECV304 cells. cDNA was coamplified by using (a) Flk-1/KDR (lane 1), Flt-1 (lane 2), or Flt-4 (lane 3); (b) FPR1 (lane 1), FPR2 (lane 2), or FPR3 (lane 3); and (c) p47phox (lane 1), p67phox (lane 2), p22phox (lane 3), or β-actin primers. PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide. (d) Serum-starved ECV304 cells were stimulated with 0.1 μM N-fMLP for the indicated times or (e) preincubated with PTX before N-fMLP stimulation for 5 minutes. Whole lysates (1 mg) were immunoprecipitated with an α-p47phox antibody and resolved on 10% SDS-PAGE. p47phox phosphorylation was determined by using an α-p-Ser antibody. An α-p47phox antibody served as a control for protein loading. (f) Superoxide production was determined as a SOD-sensitive rate reduction of cytochrome c in serum-starved ECV304 cells stimulated or not with 0.1 μM N-fMLP for the indicated times. All the experiments are representative of at least three independent experiments. compared with unstimulated cells.
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