Agonist-dependent cellular measurements of BRET between Venus-Gβ1γ2 and masGRK3ct-Rluc8 or masGRK3ct-Nluc were performed as described previously (80 (link)) to examine the activation of G protein signaling in live cells. Sixteen to twenty-four hours after transfection, HEK 293T/17 cells were washed once with phosphate-buffered saline (PBS) containing 5 mM EDTA (EDTA/PBS), and were detached by incubation in EDTA/PBS at room temperature for 5 min. Cells were harvested by centrifugation at 500g for 5 min and were resuspended in BRET buffer (PBS containing 0.5 mM MgCl2 and 0.1% glucose). Approximately 50,000 to 100,000 cells per well were distributed in 96-well flat-bottomed white microplates (Greiner Bio-One). The Rluc substrate, ViviRen (Promega), was dissolved in ethanol at a final concentration of 20 mM and stored at −20°C. ViviRen was dissolved in BRET buffer immediately before use and added to the cell suspension at a final concentration of 20 µM. The Nluc substrate furimazine was purchased from Promega and used according to the manufacturer’s instructions. BRET measurements were made with a micro plate reader (POLARstar Omega; BMG Labtech) equipped with two emission photomultiplier tubes, which enabled the detection of two emissions simultaneously with the highest possible resolution of 20 ms per data point. All measurements were performed at room temperature. The BRET signal was determined by calculating the ratio of the light emitted by Venus-Gβ1γ2 (535 nm with a 30-nm band path width) to the light emitted by masGRK3ct-Rluc8 or masGRK3ct-Nluc (475 nm with a 30-nm band path width). The average baseline value (basal BRET ratio) recorded before stimulation of cells with agonist was subtracted from the experimental BRET signal values to obtain the ΔBRET ratio.