Fig. 3. Trk and Erk 1/2 phosphorylation in trk-transfected SH-SY5Y cells. A, Western blot analysis of Trk phosphorylation status in NT-3-treated wild-type and trkC-transfected SH-SY5Y cells (clone 3:1). Serum-starved cells were incubated with 100 ng/ml NT-3 at 37°C for 10 min. After immunoprecipitation with anti-Pan-Trk antiserum and SDS-PAGE, the filter was incubated with an anti-phosphotyrosine (-PY) antibody (upper panel) and then reprobed with anti-Pan-Trk antiserum (lower panel). The approximate positions of p145trkC and p140trkA are indicated by an arrow and *, respectively. B, Western blot analysis of Trk receptor levels in SH-SY5Y/trkC cells. Serum-starved cells (clones 3:1 and 3:2) were incubated at 37°C for 10 min with 100 ng/ml NT-3 or NGF present where indicated. Cells were lysed, and cell lysates where analyzed as in A. The approximate positions of p145trkC and p140trkA are indicated by an arrow and *, respectively. C, Western blot analysis of NT-induced Trk receptor phosphorylation in trk-transfected SH-SY5Y cells. SH-SY5Y/trkC and SH-SY5Y/trkA cell lysates were analyzed as in A, with 100 ng/ml NT-3 or NGF added, where indicated. The approximate position of p145trkC is indicated by an arrow, and the p140trkA doublet is indicated by *. D, serum-starved SH-SY5Y/trkC cells (clone 3:1) were stimulated with 100 ng/ml NT-3 or NGF for 10 min and then lysed. Whole-cell lysate proteins were separated by SDS-PAGE, followed by immunoblotting with anti-phospho-Erk 1/2 antiserum and reprobing with an anti-Pan-Erk antibody. IP, immunoprecipitation; IB, immunoblot.