HEK293 cells were transiently transfected in a 10-cm dish with 1 μg of donor (RLuc8-tagged MOR WT or phosphorylation-deficient mutant) and 4 μg of acceptor (β-arrestin1–YFP, β-arrestin2–YFP, GRK2-Venus, or Rab5a-Venus). For cAMP experiments, cells were transiently transfected with the cAMP sensor using YFP-Epac-RLuc (CAMYEL) BRET biosensor (2.5 μg of FLAG-MOR and 2.5 μg of CAMYEL biosensor per dish). For GRK2 expression experiments, cells were transfected with an additional 2 μg of GRK2-WT, GRK2-DN, or pcDNA3 as a control. After 24 hours, cells were replated into poly-d-lysine–coated white opaque 96-well plates (CulturPlate, PerkinElmer) and allowed to adhere overnight. BRET experiments were performed 48 hours after transfection. Cells were washed with Hanks’ balanced salt solution (HBSS) and equilibrated in HBSS for 30 min at 37°C before the experiment. Coelenterazine h was added to a final concentration of 5 μM 10 min before dual-fluorescence/luminescence measurement in a LUMIstar Omega plate reader (BMG LabTech). The BRET signal was calculated as the ratio of light emitted at 530 nm by YFP or Venus over the light emitted at 430 nm by RLuc8. For concentration-response curves, cells were stimulated with DAMGO (10–10 to 10–4 M) or morphine (10–10 to 10–4 M) for 10 min before BRET measurements. For short kinetic experiments (β-arrestin and GRK2 recruitment), the baseline BRET ratio was measured for five cycles; then, vehicle [0.01% (v/v) dimethyl sulfoxide (DMSO)], DAMGO (1 μM), or morphine (1 μM) was added to the cells, and the BRET signal was measured for 30 min. For longer kinetic experiments (Rab5a), cells were stimulated with vehicle [0.01% (v/v) DMSO], DAMGO (1 μM), or morphine (1 μM) at different time points as stated over an interval of 100 min.

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