Immunoblot analysis of MAPK/ERK 1/2 (A) and PI3K (B) phosphorylation.

PMCs were treated with 50 ng/mL NGF (lanes 2, 3, 4, 5, and 6) or medium alone (lane 1) for 30 minutes (A) or 5 minutes (B) after pretreatment with 50 ng/mL K-252a (lane 3), 100 μmol/L PD98 059 (lane 4), 50 μmol/L LY294 002 (lane 5), or 100 μmol/L PD98 059 and 50 μmol/L LY294 002 (lane 6) inhibitors or assay medium (lane 2) for 1 hour. The phosphorylated protein products were visualized, and their relative intensity was determined. Data are representative of 5 separate experiments.