cAMP accumulation

Homogeneous time-resolved fluorescence energy transfer (HTRF) assays were performed using the Lance Ultra cAMP kit (PerkinElmer, Waltham, Massachusetts, US), based on competitive displacement of an europium chelate-labelled cAMP tracer bound to a specific antibody conjugated to acceptor beads. We first established the optimal cell density for an appropriate fluorescent signal. This was done by measuring the TR-FRET signal determined as a function of forskolin concentration using different cell densities. Forskolin dose-response curves were related to the cAMP standard curve in order to establish which cell density provides a response that covers most of the dynamic range of the cAMP standard curve. Cells were not treated or treated with vehicle or 4 μm of the indicated TAT-fused TM peptides for 4 h at 37°C in an atmosphere of 5% CO2. Cells were then grown (1,000 cells/well in white ProxiPlate 384-well microplates, PerkinElmer) in medium containing 50 μM zardaverine, pretreated at 25°C for 15 min with vehicle or the indicated receptor antagonist, stimulated with agonists for 10 min before adding 20 μM forskolin or vehicle and incubated for an additional 15-min period. Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab technologies, Offenburg, Germany).