Figure 8
8-[ϕ-575]-cAMP activates intracellular PKA. (a) For standard BRET experiments, COS-7-cells were co-transfected with the PKA type II sensor construct or negative control plasmids (bg) as indicated and grown for 48 hours. Cells were treated with the indicated amount of 8-[ϕ-575]-cAMP for 30 minutes, or mock treated (D-PBS). BRET signals were obtained after addition of the luciferase substrate DeepBlueC™ and detection of luciferase and fluorescence light emission using a multi-label reader. Shown is a representative experiment, repeated three times; data are mean ± S.E.M., performed with n = 6 replicates. (b) A BRET titration experiment was performed as described in the methods section. Briefly, cells were co-transfected with a constant amount of BRET donor (hRIIα-Rluc) and an increasing amount of acceptor DNA (GFP2-hCα) as indicated. Before BRET read-out, cells were incubated with 0.6 mM 8-[ϕ-575]-cAMP as described above. The BRET values of two independent experiments, each performed with n = 6 replicates, were background subtracted, normalized and plotted as mean ± S.E.M.