Fig. 9. NT-induced activation of Ras and phosphorylation of Erk 1/2 and Akt/PKB in SH-SY5Y/trkC and SH-SY5Y/trkA cells. A, cells were stimulated with 100 ng/ml NT-3 or 100 ng/ml NGF for 5 min. Lysates were subjected to affinity precipitation with GST-Raf-RBD. The resulting precipitates and corresponding whole-cell lysates were separated on a 10% SDS-PAGE gel. The filter with precipitated material was immunoblotted with an anti-Ras antibody. The filter with whole-cell lysate was immunoblotted with the same anti-Ras antibody, an anti-Akt/PKB, and an anti-phospho-Erk 1/2 antiserum. Arrows, an Akt/PKB protein doublet; the top band is phosporylated Akt/PKB. B, Erk 1/2 phosphorylation at increasing NT concentration. Serum-starved SH-SY5Y/trkC (clone 3:1) and SH-SY5Y/trkA cells were stimulated with increasing concentrations of NT-3 or NGF for 10 min. Whole-cell lysate proteins were separated on a 10% SDS-PAGE gel, followed by immunoblotting with anti-phospho-Erk 1/2 antiserum and reprobing with an anti-Pan-Erk antibody. IP, immunoprecipitation; IB, immunoblot.