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. 2016 Nov 3;6:36370. doi: 10.1038/srep36370

Figure 4. Localization of Epac1 at the plasma membrane is sufficient and required to inhibit neurite outgrowth.

Figure 4

(A) Representative images of N2A cells expressing Epac1-CAAX to tether Epac1 to the PM or control Epac1; scale bar corresponds to 25 μm. (B) N2A cells expressing Epac1 or Epac1-CAAX were grown in either SFM (solid bars) or RM (open bars) for 24 hrs. Data shown are the mean ± SEM neurite lengths for three independent experiments where at least 4 images of >40 cells each were analyzed for each condition. Comparison between Epac1 (SFM) and Epac1-CAAX (SFM) was performed by Student’s t-test; ***P < 0.0001. (C) Cells expressing YFP-Epac1-R82A mutant (deficient in binding phosphatidic acid at the PM) and treated with si-ctl or si-impβ1, were analyzed by live cell imaging. Representative images show that YFP-Epac1-R82A does not localize to the PM when impβ1 is knocked down; scale bar corresponds to 25 μm. Confirmation of the inability of Epac1-R82A to accumulate at the PM in response to 1 μM 8-pCPT-AM is shown in the lower left image. Western blot confirms impβ1 knockdown. (D) Cells expressing either YFP-Epac1 or YFP-Epac1-R82A mutant and treated with either si-ctl or si-impβ1 were analyzed for neurite outgrowth after culture in serum-free medium for 24 hrs. Data analyzes were performed by Two-Way ANOVA; ***P < 0.0001.