HEK293 cells were transiently transfected in suspension using Lipofectamine 2000 and seeded onto poly-d-lysine–coated white 96-well cell culture plates with solid bottom (Greiner Bio-One). Forty-eight hours after transfection, cells were washed once with Tyrode’s buffer [140 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 12 mM NaHCO3, 5.6 mM d-glucose, 0.5 mM MgCl2, 0.37 mM NaH2PO4, and 25 mM Hepes (pH 7.4)] and maintained in the same buffer. Following the addition of the luciferase substrate coelenterazine 400a, cells were either stimulated with agonist for 5 min for the G protein activity biosensor before reading BRET or stimulated and measured immediately for the DAG and PKC biosensors. BRET was measured using a Synergy Neo microplate reader (BioTek) equipped with acceptor (515 ± 30 nm) and donor (410 ± 80 nm) filters. The BRET signal was determined as the ratio of light emitted by GFP10-tagged biosensors (energy acceptors) and light emitted by RlucII-tagged biosensors (energy donors).

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