Figure 1.

Netrin-1 reduces the interaction of UNC5C with TUBB3. A, UNC5C interacts with TUBB3 in HeLa cells. TUBB3-FLAG was cotransfected with UNC5C-HA into HeLa cells. Anti-HA (UNC5C) precipitated TUBB3-FLAG. B, Netrin-1 decreases the interaction of endogenous TUBB3 with UNC5C. Dissociated neurons from P4 mouse cerebella were stimulated with either the control or netrin-1-conditioned media (netrin-1 concentration was ∼300 ng/μl) for 5 min. Cell lysates were immunoprecipitated with either anti-UNC5C (left) or with anti-TUBB3 (right), and the immunoblot was analyzed with anti-TUBB3 and anti-UNC5C. C, Quantification of B from three independent experiments. ***p < 0.001 (two-tailed Student's t test). D, E, Netrin-1 regulates the interaction of endogenous UNC5C with TUBB3 in a time-dependent (D) and dose-dependent manner (E). Primary neurons from P4 mouse cerebella were stimulated with either netrin-1-conditioned media (netrin-1 concentration was ∼300 ng/μl) for 0–20 min (D) or purified netrin-1 for 5 min (E). Bottom, Quantification. *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). F, Endogenous UNC5C interacts with TUBB3, not TUBB1 and TUBB2, in response of netrin-1 treatment. Primary neurons from P4 mouse cerebella were stimulated with either the control or netrin-1-conditioned media (netrin-1 concentration was ∼300 ng/μl) for 5 min. Cell lysates were immunoprecipitated with anti-UNC5C and followed by probing with anti-TUBB1, anti-TUBB2, or anti-TUBB3. G, Direct interaction of TUBB3 with UNC5C. Purified TUBB3 was incubated with purified intracellular domain of UNC5C tagged with GST in vitro. The anti-GST antibody was used to immunoprecipitate proteins, and the blot was analyzed with anti-TUBB3.