Figure 9. Representative analysis for binding kinetic of propranolol to transiently expressed HiBiT-β₂-AR in the presence of NanoBRET GPCR Tracer G-3.

HEK293 cells expressing HiBiT-β₂-AR were assayed as indicated in the protocol. Briefly, cells were transfected, grown overnight, and next day treated with HiBiT detection reagent for 15 min prior to the treatment with propranolol titration alongside a fixed EC₈₀ concentration of NanoBRET GPCR Tracer G-3 (i.e., the previously described propranolol-BODIPY ( Boursier et al., 2020 )). Kinetic measurements of filtered luminescence were taken using GloMax Discover Microplate Reader (Promega) equipped with a 450 nm (8-nm bandpass) filter (donor) and a 600-nm long pass filter (acceptor) (n = 3; Error bars indicate SD).