Figure 2
From: Epac1 interacts with importin β1 and controls neurite outgrowth independently of cAMP and Rap1
Impβ1 serves as a cytosolic anchor for Epac1.
(A) N2A cells co-expressing YFP-Epac1 (green) and mcherry-impβ1 (red) were treated with either vehicle or 1 μM 8-pCPT-AM for 15 min, followed by live cell imaging. Representative images of >50 cells in each condition are shown; scale bar corresponds to 25 μm. (B) N2A cells expressing YFP-Epac1 or GFP were treated with either vehicle or 10 μM 8-pCPT-AM for 15 min. Epac1 was pulled down using the GFP-TRAP beads and samples were analyzed by western blotting with impβ1 and Epac1 antibodies. (C) N2A cells overexpressing YFP-Epac1 were treated with impβ1 si-RNA (si-impβ1) or control si-RNA (si-ctl) and the effect on Epac1 localization was monitored by live cell imaging. Images are representative of at least three independent experiments with >50 cells each; scale bar corresponds to 25 μm. Western blot shows confirmation of impβ1 knockdown. (D) PM sheets of control (ctl) EA.hy926 cells or EA.hy926 cells treated with 1 μM 8-pCPT-AM for 15 min (left panels), or EA.hy926 cells treated with si-impβ1 or si-ctl (right panels) were attached to EM grids, immunolabeled with gold nanoparticles coupled to Epac1 antibody (H-70), and imaged by EM. Images are representative of at least 15 PM sheets per condition; scale bar corresponds to 200 nm. Quantification of the number of gold-coupled Epac1 nanoparticles per μm2 area in the inner leaflet of the plasma membrane is shown in the bar graphs. Data were analyzed by Student’s t-test; ***P < 0.001.
