FIG. 1.

A role for the PI3K-Akt pathway in endothelial cell migration in response to Semaphorin 4D. (A) PI3K inhibition prevents Semaphorin 4D-mediated endothelial cell migration. Cells were preincubated with either DMSO vehicle control (−) or 50 μM of the PI3K inhibitor LY294002 (+) and used in a chemotaxis assay with purified (200 ng/ml) Semaphorin 4D as the chemoattractant (S4D). Media containing 10% fetal bovine serum (S) were used as positive controls for migration. The bars represent the fold increase of migration as determined by densitometry relative to that seen in negative control wells containing 0.1% BSA (C). (B) Endothelial cells were treated with 200 ng/ml Semaphorin 4D (S4D) for the indicated periods of time, lysed, and immunoprecipitated for the PI3K subunit p85 (IP: p85) and then immunoblotted for the presence of phosphorylated tyrosine residues (WB: P-Y). Phosphotyrosine was seen in p85 immunoprecipitates from Semaphorin 4D-treated cells starting at 1 min after treatment (upper panel). Total p85 was used as a loading control (WB: p85; lower panel). (C) Semaphorin 4D activates Akt in endothelial cells. Cells were treated with 200 ng/ml Semaphorin 4D (S4D) for the indicated periods of time, lysed, and analyzed for the presence of phosphorylated Akt. Phospho-Akt (P-Akt, Thr308) was seen in cells starting at 1 min after treatment (upper panel). Total Akt was used as a loading control (WB: Akt; lower panel). (D) Semaphorin 4D-mediated activation of Akt can be inhibited by LY294002 in endothelial cells. Cells preincubated with either vehicle control (C) or 50 μM LY294002 (LY) were treated with 200 ng/ml Semaphorin 4D (S4D) for the indicated periods of time, lysed, and analyzed for the presence of phosphorylated Akt. Phospho-Akt (P-Akt, Thr308) was seen in cells starting at 1 min after treatment in DMSO-pretreated cells but not in those treated with LY294002 (upper panel). Total Akt was used as a loading control (WB: Akt; lower panel).