Figure 5.

Epac-selective cAMP analogs fail to activate the PG gene promoter. A, expression of endogenous 110 kDa Epac2 in GLUTag cells was confirmed by Western blot analysis using an anti-Epac2 5B1 MAB. Epac2 immunoreactivity was also detected in a lysate from WT mouse brain but not a negative control lysate from an Epac2 knockout (KO) mouse brain (72). B, −2400 Glu-Luc activity was not stimulated after a 4-hour exposure of GLUTag cells to the non-AM esters (left) or AM-esters (right) of Epac-selective cAMP analogs. As a positive control, the responsiveness of −2400 Glu-Luc was confirmed using GLUTag cells treated with non-AM esters of cAMP analogs that activate PKA (middle). C, the efficacy of Epac-selective cAMP analog 8-pCPT-2′-O-Me-cAMP-AM was validated in a pull-down Rap1 activation assay using lysates of GLUTag cells transfected with FLAG-Rap1. Levels of active Rap1-GTP (C₁) and total Rap1 (C₂) were determined by Western blot analysis using an anti-FLAG MAB.