Transfected HEK293T Cell Signaling Assay

Signaling experiments were performed on whole cells with transiently transfected HEK293T cells as described before^{11 (link),14 (link)}. Twenty-four hours after transfection, cells were washed with PBS, detached with cell dissociation buffer (Gibco) and washed again in PBS. Cells were resuspended in assay buffer [10 mM Hepes (pH 7.4), 146 mM NaCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 4.2 mM KCl, 5.5 mM glucose, 50 mM LiCl, 1 mM 3-isobutyl-1-methylxanthin). cAMP and IP1 accumulation assays were performed on white low-volume 384-well plates (Greiner) using the cAMP Tb kit and the IP-One Tb kit (both from CisBio), respectively, according to the manufacturer’s protocol. For cAMP accumulation, 5000 cells were incubated with agonist at the indicated concentrations for 30 min at RT. To determine basal receptor signaling, cells were incubated in isobutylmethylxanthine (IBMX)-containing assay buffer for 30 min in the absence of ligand. For IP1 accumulation, 20,000 cells were incubated with agonist at the indicated concentrations for 2 h at 37 °C. Fluorescence intensities were measured on a Spark fluorescence plate reader (Tecan). To generate concentration-response curves, data were fitted to a three-parameter logistic equation.