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Variations of bioluminescence resonance energy transfer (BRET) assay were performed to detect receptor ligand-induced events. A constant amount of plasmid cDNA (15 μg) was transfected into human embryonic kidney cells 293 T (HEK-293T) using polyethylenimine (PEI; Sigma) in a 1:2 weight ratio in 10 cm plates. Cells were maintained in culture with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Atlanta), 2 mM-glutamine (Gibco), and 1% penicillin streptomycin (Gibco) and kept in an incubator at 37 °C and 5% CO. The transfected amount and ratio among the receptor and heterotrimeric G proteins were tested for the optimized dynamic range in drug-induced BRET. Experiments were performed approximately 48 h post-transfection. As reported previously, (50) cells were collected, washed, and resuspended in phosphate-buffered saline (PBS). Approximately 200 000 cells/well were distributed in 96-well plates, and 5 μM coelenterazine H (luciferase substrate, BRET1) or 5 μM coelenterazine 400a (luciferase substrate, BRET2) was added to each well. One minute after the addition of coelenterazine, D2R ligands were added to each well. Four different configurations of BRET were used: (i) Gαi1-γ2 protein activation, (ii) GαoA-γ2 protein activation, (iii) cAMP inhibition, and (iv) β-arrestin-2 recruitment. (i) Gαi1-γ2 protein activation assay uses Gαi1-Rluc-γ2-GFP10 for a resonance energy transfer (RET) pair. D2R and untagged Gβ1 constructs were cotransfected; (ii) GαoA-γ2 protein activation assay uses GαoA-Rluc-γ2-GFP10 for a RET pair. D2R and untagged Gβ1 constructs were cotransfected; (iii) cAMP production assay uses a CAMYEL biosensor construct that contains Rluc and YFP allowing detection of intracellular cAMP change (59) in conjunction with receptor coexpression. D2R-Gi/o activation was studied by agonist-induced inhibition of cAMP production. Cells were prestimulated with 10 μM forskolin (Sigma) 10 min prior to agonist treatment. (iv) β-Arrestin-2 recruitment uses D2R-Rluc-β-arrestin-2-Venus for a RET pair. GRK2 was cotransfected to assist an enhanced phosphorylation required for the β-arrestin-2 recruitment. The donor luminescence as well as the acceptor fluorescence was always quantified for consistent expression levels across different experiments such that no significant expression differences between Gαi1-Rluc-γ2-GFP10 and GαoA-Rluc-γ2-GFP10 pairs were observed, for instance. For BRET1, venus was excited at 500 nm and measured at an emission wavelength of 530 nm. For BRET2, GFP10 was excited at 405 nm and measured at an emission wavelength of 515 nm. Both fluorophores were measured over 1 s of recording, using a Mithras LB940 microplate reader (Berthold Technologies, Bad Wildbad, Germany). The BRET1 signal from the same batch of cells was calculated as the ratio of the light emitted by Venus (530 nm) over that emitted by coelenterazine H (485 nm), and the BRET2 signal from the same batch of cells was calculated as the ratio of the light emitted by GFP10 (515 nm) over that emitted by coelenterazine 400a (400 nm). BRET change was defined as BRET ratio for the corresponding drug minus BRET ratio in the absence of the drug.values are expressed as the basal subtracted BRET change and in the dose–response graphs. Data and statistical analysis were performed with Prism 7 (GraphPad Software).