FIG. 6.

PI 3-kinase activity does not contribute to the activation of ERKs after NGF stimulation. (A) PC12 cells were pretreated (+) or not pretreated (−) for 30 min with the indicated concentrations of the PI 3-kinase inhibitor LY294002 or with vehicle (Me₂SO) and then stimulated (+) or not stimulated (−) for 5 min with NGF. After treatment, cells were lysed and protein extracts were analyzed by Western blotting with an anti-phospho-Akt antibody (upper panel) or an anti-phospho-ERK antibody (middle panel) and stripped and reprobed with an anti-pan-ERK antibody (lower panel) as a control for the protein content per lane. (B) PC12 cells were stimulated (+) or not stimulated (−) for 1 min with NGF. After treatment, cells were lysed and protein extracts were subjected to immunoprecipitation with the anti-Tyr(P) antibody 4G10. PI 3-kinase activity was assayed in the immunoprecipitates in the presence (+) or in the absence (−) of 10 μM LY294002. Arrows labeled PI 3-P³² and Origin indicate the positions of in vitro radiolabeled l-α-phosphatidylinositol and the sample application, respectively.