Lgbit stock solution

Parental HEK 293 cells, G_12/13-deficient HEK 293 cells (39 (link)), or β-arrestin1/2-deficient HEK 293 cells (37 (link)) in growth phase were seeded in a 6-well culture plate at a concentration of 2 × 10⁵ cells mL⁻¹. Cells were transfected with 100 ng of HiBiT-V₂R, which contained an Interleukin 6-derived signal sequence followed by a HiBiT sequence and a linker at the N terminus (MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVSGWRLFKKISGGSGGGGSG; gene synthesized with codon optimization) and an unintended SmBiT tag at the C terminus. After 1 d, cells were harvested, suspended in 1 mL of assay buffer, dispensed in a white 96-well half-area plate at a volume of 25 µL per well, and mixed with 25 µL of 2× substrate buffer consisting of 1:200 of a LgBiT stock solution (Promega) and 20 µM furimazine in the assay buffer. After 40 min at room temperature, the plate was measured for baseline luminescence, and a titrated ligand (10 µL) diluted in the 1× substrate buffer was manually added. The plate was immediately read at room temperature for the following 30 min at a measurement interval of 30 s with an accumulation time of 0.4 s per read. The luminescence counts over 27–30 min after ligand addition were averaged and normalized to the initial count.