Figure 7: RapGEF2 phosphorylation controls neuronal migration via Rap1.

(a) Pull-down assay of active Rap1 in the lysates of HEK293T cells expressing vector control (Con), wild type (WT), phosphodeficient (S1124A) or phosphomimetic (S1124E) mutants of RapGEF2 using GST-RalGDS-RBD. (b) Quantification of fold change of active Rap1 levels. Error bars indicate the s.e.m. of four independent experiments. *P<0.05 versus WT-expressing group; Student’s t-test. (c) Pull-down assay of active Rap1 in the cortical lysates from E18 Cdk5^−/− mice and their corresponding wild-type littermates using GST-RalGDS-RBD. See full-length blots in Supplementary Fig. 10. (d) Quantification of fold change of active Rap1 levels. Error bars indicate the s.e.m. of three independent experiments. (e) Co-electroporation of E14 mouse brains was performed using RapGEF2 shRNA (shRapGEF2) together with control (pCAG) or the indicated RapGEF2-expressing plasmids (WT, S1124A or S1124E). E17 coronal cortical sections were stained for GFP and TO-PRO3. Scale bar, 100 μm. (f) Quantification of the percentages of GFP⁺ cells expressing control or different RapGEF2 constructs in different cortical layers at E17. Error bars indicate the s.e.m. of four different brains containing >600 neurons. (g) Quantification of the percentages of GFP⁺ neurons with uni- or bipolar morphology, no process and multiple (≥3) processes. Error bars indicate the s.e.m. of three different brains containing >120 neurons. *P<0.05, **P<0.01, ***P<0.001 versus shRapGEF2+pCAG group; Student’s t-test.