NanoBiT-G-protein dissociation assay

G-protein activation was detected by a NanoBiT-G-protein dissociation assay⁶⁹. In brief, HEK293T cells were plated in each well of a six-well plate at a concentration of 0.3 million/ml (2 mL per well). Plasmid transfection was performed with a mixture of 400 ng Gαio-LgBiT, 500 ng Gγ-SmBiT, 500 ng Gβ, 600 ng ghrelin receptor by Lipofectamine 2000 (ThermoFisher Scientific) in 200 μL of Opti-MEM (Gibco). After 1 day of transfection, cells in the six-well plate were digested and resuspended in complete medium DMEM (5% FBS, 1% antibiotic) and plated in a 96-well flat-bottomed white microplate. After 24 h, the cells were washed twice with D-PBS and incubated in 40 μL of 5 μM coelenterazine 400a (Maokangbio) solution diluted 0.01% BSA and 5 mM HEPES (pH 7.5)-containing HBSS (assay buffer) for 1 h at room temperature. Baseline luminescence was measured using a luminescent microplate reader (Tecan). Ghrelin (5×, diluted in the assay buffer) was added to the cells (10 μL) and incubated for 3–5 min at room temperature before the second measurement. The ligand-induced signal ratio was normalized to the baseline luminescence and fold-change signals over vehicle treatment were used to show G-protein dissociation response.