Figure 1.

8-NBD-cAMP Competition Assay Using Purified EPAC1-CNB and EPAC2-CNB Proteins. (a)The 8-NBD-cAMP competition assay was carried out as previously described¹⁹. Briefly, purified EPAC1-CNB (0.8 µM), EPAC2-CNB (0.8 µM), GST (0.8 µM) or assay buffer (no protein) was incubated in the presence of cAMP (50 µM) or DMSO. 8-NBD-cAMP (0.1 µM) was then added to every well and the fluorescence intensity was measured after four hours. Significant inhibition of fluorescence in the presence of cAMP is indicated, ^#P < 0.0001 (n = 3). (b) Inhibition of 8-NBD-cAMP (62.5 nM) fluorescence, resulting from interaction with EPAC1-CNB (0.8 µM), is plotted in the presence of varying concentrations of known competitor compounds; cAMP (red squares), 007 (blue circles) or 8-CPT (green triangles). IC50 values were calculated for each competition assay and are shown in comparison with published Kd values for each competitor.