Concentration-response curves and FRET experiments set up.

a, Cells were stimulated with UCN1 (upper) or UCN3 (bottom) at the indicated concentration for 6 minutes. pERK1/2 and total ERK1/2 were determined by western blot and quantified by densitometry analysis. pERK1/2 was normalized total ERK1/2 in each concentration point. Results are expressed as the percentage of maximum pERK1/2 obtained. Data: Mean ± SEM from 3 independent experiments. To determine EC50 for UCNs-induced ERK1/2 phosphorylation, data were adjusted with 3-parameter dose-response curve (EC50_UCN1: 0,43 nM, EC50_UCN3: 0,041 nM). b, Cells were transfected with FRET biosensors EPAC-S^H187 stimulated with 10 and 100 nM UCN1. The curves illustrate the cAMP response over a 25-minute time frame. Data: Mean +/- SEM, n = 5–10 cells.

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