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. 2020 Sep 2;6(36):eaba1693. doi: 10.1126/sciadv.aba1693

Fig. 5. DSCAM associates with RapGEF2 and prevents Rap1 activation.

Fig. 5

(A) Diagram of the domain structure of RapGEF2 deletion constructs. (B) Coimmunoprecipitation assay revealed that DSCAM associates with the RapGEF2 cNMP and Nter domains. Lysates of COS-7 cells expressing RapGEF2 constructs described in (A) and DSCAM were incubated with an anti-GFP antibody. The input (bottom column, 5%) and immunoprecipitants (upper two columns) were analyzed by Western blotting using anti-DSCAM and anti-GFP antibodies. The experiment was repeated at least three times. (C) Pull-down assay to evaluate active Rap1 using COS-7 cells expressing control EGFP or EGFP-RapGEF2 with or without Flag-Dscam cytoplasmic region (Flag-DSCAM-C1; described in Fig. 4F). The input (bottom four columns, 5%) and immunoprecipitants (upper column) were analyzed by Western blotting using anti-Rap1, anti-GFP, and anti-Flag antibodies. (D) Quantification of fold change of active Rap1 levels. Normalized active Rap1 activity was calculated by active Rap1/total Rap1. Data are presented as the mean ± SEM from three independent experiments; unpaired two-tailed t test.