A, SH-SY5Y-TrkB cells were transiently transfected with eukaryotic expression vectors of PTP1B (WT, C215S PTP1B, or parental pMT2 vector) using the PEI method. Cells were stimulated with BDNF (25 ng/ml) or vehicle (veh; PBS) for 5 min. Representative anti-phospho-TrkB, anti-phospho-AKT, anti-phospho-ERK, anti-TrkB, anti-AKT, anti-ERK, anti-human PTP1B, and anti-SHP2 immunoblots of total lysates are shown and quantified in B. Data are represented as mean ± S.E. (n = 3; *, p < 0.05 compared with empty vector group, Student's t test). C and D, lysates were immunoblotted with anti-phospho-TrkB and anti-SHP2. BDNF dose for each time point in C is 25 ng/ml. Time point for each dose in D is 5 min. Graphs show quantified results from at least three separate experiments, and representative blots are shown. Dashed lines show where the image from the same blot was cropped. Data are represented as mean ± S.E. (n = 3; *, p < 0.05 compared with empty vector group, two-way ANOVA with post hoc Bonferroni test).

A, SH-SY5Y-TrkB cells were pretreated with cell permeable PTP1B-specific inhibitor (compound II (100 nm)) or vehicle (veh; dimethyl sulfoxide) for 5 min prior to stimulation with BDNF (25 ng/ml) or vehicle (PBS) for 5 min. Representative anti-phospho-TrkB, anti-phospho-AKT, anti-phospho-ERK, anti-TrkB, anti-AKT, anti-ERK, and anti-SHP2 immunoblots of total lysates are shown and quantified in B. Data are represented as mean ± S.E. (n = 3; *, p < 0.05 compared with control group, Student's t test). C and D, lysates were immunoblotted with anti-phosphoTrkB and anti-SHP2. BDNF dose for each time point in C is 25 ng/ml; time point for each dose in D is 5 min. Graphs show quantified results from at least three separate experiments and representative blots are shown. Dashed lines show where the image from the same blot was cropped. Data are represented as mean ± S.E. (n = 3; *, p < 0.05 compared with control group, two-way ANOVA with post hoc Bonferroni test).

A, hypothalamic TrkB phosphorylation in 15-week-old male Ptpn1^−/− mice and wild-type littermates after 12 weeks of high-fat diet feeding. Representative immunoblots of lysates using anti-phospho-TrkB, anti-TrkB, anti-mouse PTP1B, and anti-SHP2 antibodies are shown. Data are represented as mean ± S.E. (n = 4 for wild-type and n = 3 for Ptpn1^−/−; *, p < 0.05 compared with wild-type group, Student's t test). B, hippocampal TrkB phosphorylation in 15-week-old, male Ptpn1^−/− mice and wild-type littermates after 12 weeks of high-fat diet feeding. Dashed lines show where the image from the same blot was cropped. Representative immunoblots of lysates using anti-phosphoTrkB, anti-TrkB, anti-mouse PTP1B, and anti-SHP2 antibodies are shown. Data are represented as mean ± S.E. (n = 3 for each group; #, p = 0.067, Student's t test).

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.

A, cumulative chow intake (in grams) of 4–5-month-old Ptpn1^−/− male mice and wild-type littermates at 1, 3, and 6 h following BDNF (0. 2 μg) or aCSF (vehicle) infusion into the lateral ventricle. Data are represented as mean ± S.E. (n = 10 for wild-type, n = 5 for Ptpn1^−/− mice; *, p ≤ 0.05; #, p = 0.066 compared with vehicle treatment, one-way ANOVA). B, 24 h % body weight suppression (compared with vehicle treatment) of 4–5-month-old Ptpn1^−/− male mice and wild-type littermates following BDNF (0.2 μg) or aCSF (vehicle) infusion into the lateral ventricle. Data are represented as mean ± S.E. (n = 10 for wild-type; n = 5 for Ptpn1^−/− mice; *, p < 0.05 compared with wild-type controls, Student's t test). C, core temperature of 4–5-month-old Ptpn1^−/− male mice and wild-type littermates following BDNF (0.05 μg) or aCSF (vehicle) infusion into the lateral ventricle. Data are represented as mean ± S.E. (n = 10 for wild-type; n = 5 for Ptpn1^−/− mice; *, p ≤ 0.05 compared with vehicle treatment; **, p < 0.05 compared with wild-type controls, one-way ANOVA).

A, representative image of SH-SY5Y-TrkB cells with vehicle (Veh)/BDNF treatment (left) or PTP1B inhibitor/BDNF treatment (right) at 6 h post-stimulation (t = 6). Arrows point at primary neurites (scale bar, 80 μm). B, SH-SY5Y-TrkB cells were pretreated with cell permeable, PTP1B-specific inhibitor (compound II (500 nm)) or vehicle (dimethyl sulfoxide) for 5 min prior to stimulation with BDNF (25 ng/ml) or vehicle (PBS). Primary neurite outgrowth was assessed prior to and 2, 4, 6, and 24 h following BDNF or vehicle stimulation. Average primary neurite length was quantified using ImageJ software (Neuron J plugin). Data are represented as mean ± S.E. (five neurites in a representative field were quantified and averaged for each time point, and the experiment was repeated three times; *, p < 0.05 compared with respective vehicle controls and **, p < 0.05 PTP1B inhibitor/BDNF treatment compared with vehicle/BDNF treatment, one-way ANOVA).