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. Author manuscript; available in PMC: 2019 Mar 15.
Published in final edited form as: Neuroscience. 2018 Jan 31;374:155–171. doi: 10.1016/j.neuroscience.2018.01.046

Figure 3.

Figure 3

TUBB3 mutations inhibit netrin-1-induced interaction of DCC with TUBB3. A–D. TUBB3 shRNAs were co-transfected with wild type human TUBB3 (A–D), R262C (A and B) or A302V (C and D) into mouse E15 cortical neurons. E–F. Mouse E15 cortical neurons were transfected with either wild type human TUBB3 or A302V. Primary neurons were cultured overnight and stimulated with purified netrin-1 or sham-purified control for 20 min. Cell lysates were immunoprecipitated with anti-DCC and followed by probing with anti-DCC, anti-FLAG antibodies. B, D and F. Quantification of relative binding of DCC to TUBB3-FLAG (B, D and F), R262C-FLAG (B) and A302V-FLAG (D and F). The Y axis is the normalized ratio of mean intensity (arbitrary units) of FLAG to DCC. Data are mean ± s.e.m from three separate experiments. *p<0.05, **p<0.01, ***p<0.0001, ns indicates no significant difference (one-way Anova and Tukey’s test for post-hoc comparsions).