FIG. 2.

NGF treatment of endothelial cells stably expressing Trk-A/Plexin-B1 chimeric receptor constructs induces Akt phosphorylation. (A) Cells stably expressing extracellular Trk-A or Trk-A fused to the intracellular portion of Plexin-B1 (Trk-A/Plexin-B1) were treated with 100 ng/ml NGF for the indicated periods of time, lysed, and analyzed for the presence of phosphorylated Akt (P-Akt, Thr308; upper panels). Cells treated with 200 ng/ml Semaphorin 4D for 5 min were used as a positive control (S4D; lane 1). Total Akt was used as a loading control (WB: Akt; lower panels). (B) NGF-induced phosphorylation of Akt in endothelial cells stably expressing Trk-A/Plexin-B1 chimeric receptor constructs can be inhibited by LY294002. Cells stably expressing Trk-A/Plexin-B1 chimeric receptors were preincubated with either 50 μM LY294002 (LY) or DMSO vehicle control (C) and treated with 100 ng/ml NGF for the indicated periods of time, lysed, and analyzed for the presence of phosphorylated Akt. Phospho-Akt (P-Akt, Thr308) was seen after treatment in control pretreated cells (lanes 7 to 9, upper panel) but not in those growing in LY294002 (lanes 4 to 6, upper panel). Cells treated with 200 ng/ml Semaphorin 4D and DMSO (C) or 100 nM Wortmannin (W) for 5 min were used as a positive control (lane 1) and negative control (lane 2), respectively. Total Akt was used as a loading control (WB: Akt, lower panel).