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. 2010 Jun 14;30(16):3956–3969. doi: 10.1128/MCB.00242-10

FIG. 8.

FIG. 8.

Attenuation of Rap1 activation at the NE by depletion of endogenous Epac1. (A) Depletion of Epac1 by siRNA. (Top) Lysates of HEK293 cells transfected with scrambled (S) siRNA or Epac1 siRNA were examined by Western blotting using anti-Epac1 and anti-ERK2 (loading control). (Bottom) Quantification of the results of three experiments (means ± standard errors of the means; *, P < 0.05). (B) Specificity of siRNA for Epac1. HEK293 cells were transfected with Flag-Epac1 or Flag-Epac2 cDNA and either scrambled or Epac1 siRNA, as indicated. The expression of transfected Epac isoforms in the lysates of transfected cells was examined by Western blotting using anti-Flag antibodies. Flag-ERK2 served as a transfection and loading control. (C) Effect of Epac1 depletion on Rap1 activation at the NE. GFP-RBDRalGDS and mCherry-Rap1b were cotransfected with scrambled or Epac1 siRNA in HEK293 cells. The percentages of cells with enrichment of GFP-RBDRalGDS on the NE were quantified from four independent experiments (means ± standard errors of the means; *, P < 0.01). (D) Representative images from the experiment for which results are shown in panel C. The top and bottom panels show cells cotransfected with scrambled and Epac1 siRNA, respectively. (Left) GFP; (center) mCherry; (right) merged images. Bar, 10 μm. (E) Effect of Epac1 depletion on Rap2 activation. GFP-RBDRalGDS and mCherry-Rap2b were cotransfected with scrambled (top) or Epac1 (bottom) siRNA in HEK293 cells. (Left) GFP; (center) mCherry; (right) merged images. Bar, 10 μm. (F) Model for anchored Epac1 signaling at the NE. The NPC is a large multimeric protein complex, shown in cross section, spanning the double bilayer of the NE. Epac1 is localized to the NPC through direct interaction between its RA domain and Ran-GTP, which also associates with RanBP2, the major nucleoporin on the cytoplasmic face of the NPC. Epac1 may also make direct contacts with RanBP2 as shown (question mark). The anchored Epac1 allows cAMP to activate local pools of Rap1 that are tethered on the NE.