NT-3 promotes TrkC binding to PTPσ to trigger TrkC-mediated presynaptic differentiation. A, Saturation binding of Ig-PTPσ to HA-TrkC expressed in HEK293T cells in the absence or presence of NT-3 (25 or 100 ng/ml). K_ds were calculated from three independent experiments. All data are shown as means ± SEM. B, Representative images of the heterologous synapse-formation activities of TrkC. Neurons were cocultured for 2 d with HEK293T cells expressing EGFP alone (Control) or HA-TrkC (TrkC) in the absence (+DMSO) or presence of 25 ng/ml NT-3, and then stained with antibodies against EGFP or HA (blue) and synapsin (red). Scale bar, 10 μm (applies to all images). C, Synapse-forming activity in B was quantified by measuring the ratio of synapsin staining intensity (red) to HA immunoreactivity intensity (blue). All data are shown as means ± SEM (³*p < 0.001; ANOVA with post hoc Tukey's test). D, E, Same as B and C except that the NT-3 or BDNF concentration was 100 ng/ml (³*p < 0.001; ANOVA with post hoc Tukey's test). Scale bar, 10 μm (applies to all images). F, G, Representative images (F) and quantification results (G) demonstrating no alteration in dendritic branch number in NT-3-treated cultured neurons. Hippocampal neurons at DIV7 were treated at the indicated concentration of NT-3 and analyzed by Sholl analysis at DIV10. Scale bar, 60 μm (applies to all images). All data are shown as means ± SEM. n denotes the number of neurons as follows: Control, n = 20; NT-3 (25 ng/ml), n = 21; and NT-3 (100 ng/ml), n = 21.