Figure 3.

Netrin-1 decreases the interaction of UNC5C with polymerized TUBB3. A, Taxol and nocodazole (Noc) inhibited the netrin-1-induced UNC5C/TUBB3 dissociation. Dissociated P4 mouse cerebellar neurons were treated with purified netrin-1 in the presence of 1 μm Taxol, 3 μm nocodazole, or DMSO. B, Quantification of A from three independent experiments showing relative binding of UNC5C to TUBB3. ***p < 0.001 (one-way ANOVA and Tukey's test for post hoc comparisons). C, Primary P4 mouse cerebellar neurons were stimulated with netrin-1 or sham-purified control, and a cosedimentation assay of cell lysates was performed in the absence or presence of Taxol. UNC5C and TUBB3 in the pellet (P) and supernatant (S) fractions were examined by immunoblotting using anti-UNC5C and anti-TUBB3 antibodies, respectively. D, Quantification of C from three independent experiments showing P/S ratio of UNC5C and TUBB3. *p < 0.05 (one-way ANOVA and Tukey's test for post hoc comparisons). ***p < 0.001 (one-way ANOVA and Tukey's test for post hoc comparisons). E, Dissociated P4 mouse cerebellar neurons were transfected with either TUBB3 shRNA or control shRNA and stimulated with purified netrin-1. The cosedimentation assay was conducted with Taxol to stabilize MTs in vitro as above. F, Quantification of P/S ratio of E from three independent experiments. ***p < 0.001 (two-tailed Student's t test). G, Knockdown of endogenous UNC5C in primary neurons. P4 mouse cerebellar neurons were nucleofected with control shRNA, UNC5C shRNA, or UNC5C shRNA plus wild-type human UNC5C. Ctl, Control shRNA; R, wild-type human UNC5C. H, TUBB3 shRNA specifically knocked down endogenous TUBB3. P4 mouse cerebellar neurons were transfected with control shRNA, TUBB3 shRNA, or TUBB3 plus wild-type human TUBB3. Ctl, Control shRNA; R, wild-type human TUBB3.