A, purified, active, recombinant GST fusion proteins (GST only control, WT, and D181A/Y46F PTP1B) (10 μg) were incubated with BDNF-stimulated (50 ng/ml, 5 min) (left panel, lanes 1–3, respectively) or unstimulated (right panel, lanes 1–3, respectively) SH-SY5Y-TrkB cell lysates (1. 5 mg) in a substrate-trapping (GST pulldown) experiment. Additionally, D181A/Y46F PTP1B (10 μg) was incubated with BDNF-stimulated (50 ng/ml, 5 min) SH-SY5Y-TrkB cell lysate (1.5 mg) in the presence of sodium orthovanadate (left panel, lane 4) and with BDNF-stimulated (50 ng/ml, 5 min) kinase-dead SH-SY5Y-TrkB cell lysates (Y702F/Y706F/Y707F TrkB and Y706F/Y707F TrkB (left panel, lanes 5 and 6, respectively)) (1.5 mg). Protein complexes were pulled down with glutathione beads and immunoblotted with anti-phospho-TrkB, anti-TrkB, and anti-human PTP1B as indicated. Lysates used as input were separately immunoblotted with anti-phospho-TrkB and anti-TrkB. B, purified, active, recombinant GST fusion proteins (GST-only control, WT and D181A/Y46F PTP1B) (10 μg) were incubated with BDNF-stimulated (0.5 μg, 45 min) mouse brain tissue lysate (3 mg) in a substrate-trapping (GST pulldown) experiment. Protein complexes were pulled down with glutathione beads and immunoblotted with anti-TrkB and anti-GST antibodies, as indicated.