FIG. 5.

Blockade of Src prevents Semaphorin 4D-induced Akt activation and cell migration. (A) PP2 blocks Src and Akt phosphorylation in Semaphorin 4D-treated endothelial cells. Endothelial cells were preincubated with either 10 μM PP2 (PP2) or DMSO (C) and treated with 200 ng/ml Semaphorin 4D for the indicated periods of time, lysed, and analyzed for the presence of phosphorylated Akt and Src. Phospho-Src (WB: P-Src) was seen after 1 min of treatment in cells preincubated with DMSO but not in cells grown in PP2 (upper panels). Total Src was used as a loading control (WB: Src). Phosphorylation of Akt is also seen in DMSO-treated control cells but not in PP2 treated populations [WB: P-Akt (Thr 308), lower panels]. Total Akt is used as a loading control (WB: Akt). (B) PP2 blocks Semaphorin 4D/Plexin-B1-mediated chemotaxis. Endothelial cells (control) or cells stably expressing Trk-A/Plexin-B1 chimeric receptors (Trk-A/PB1) were preincubated with either DMSO vehicle control (C) or 10 μM of the Src inhibitor PP2 (PP2) and then used in a cell migration assay with Semaphorin 4D (S4D) or 100 ng/ml NGF (NGF) as the chemoattractants. 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).