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

Figure 5. Knockdown of impβ1 promotes membrane accumulation of Epac1 and inhibition of neurite outgrowth independently of cAMP binding.

Figure 5

(A) N2A cells overexpressing the Epac1 FRET reporter were treated with si-ctl or si-impβ1 and analyzed by flow cytometry following treatment with either vehicle or 30 μM 8-pCPT-AM for 15 min. Bar graph shows the percentage of the FRET ratio (405/530) in YFP-positive cells, in si-ctl (open bars) as compared to si-impβ1 (solid bars) samples. (B) N2A cells overexpressing YFP-Epac1 were treated with si-ctl or si-impβ1, followed by treatment with vehicle or 0.1 μM 8-pCPT-AM for 15 min. Rap1-GTP was pulled down with Ral GDS-Rap binding domain beads followed by western blotting to detect Rap1-GTP and total Rap1, impβ1, and Epac1. (C) Binding of purified Epac1 to membrane lipid strips in the absence or presence of 50 μM 8-pCPT for 15 min. (D) Representative images of N2A cells overexpressing YFP-Epac1-R279L mutant (deficient in binding cAMP) and treated with si-ctl or si-impβ1 were analyzed by live cell imaging. Representative images show that YFP-Epac1-R279L localizes to the PM when impβ1 is knocked down. Lower left image confirms that Epac1-R279L does not respond to stimulation with 8-pCPT-AM. (E) N2A cells overexpressing either YFP-Epac1-R279L mutant or YFP-Epac1 and treated with si-ctl or si-impβ1, were grown in SFM (solid bars) or RM (open bars). Neurite length was analyzed as described in the legend to Fig. 3. (F) The Epac inhibitor ESI-09 does not prevent the effect of impβ1 depletion on PM accumulation of Epac1. N2A cells overexpressing YFP-Epac1 were treated with si-ctl or si-impβ1 and 5 μM ESI-09. Representative images showing that YFP-Epac1 also localizes to the PM in the presence of ESI-09 when impβ1 is knocked down. (G) N2A cells overexpressing YFP-Epac1 treated with si-ctl or si-impβ1, were grown in SFM supplemented with vehicle (open bars) or 5 μM ESI-09 (solid bars) for 24 hrs. Data in all panels were analyzed by Two-Way ANOVA; ns: not significant, ***P < 0.001, and **P < 0.01. Scale bar in all images corresponds to 25 μm.