Fig 4. BRET indicates 5-HT_2AR:5-HT_2CR interaction within 10 nm.

(A) Schematic of BRET assay between 5-HT_2CR-RLuc and 5-HT_2AR-eYFP constructs. (B) Representative concentration response curve for 5-HT-mediated intracellular calcium (Ca_i²⁺) release for WT 5-HT_2AR (red) and 5-HT_2AR-RLuc (purple) constructs transiently transfected in HEK293 cells. The 5-HT-mediated increase in Ca_i²⁺ dye fluorescence over baseline was calculated as the % maximal response of WT 5-HT_2AR. (C) Representative concentration response curve for 5-HT-mediated Ca_i²⁺ release for WT 5-HT_2CR (blue) and 5-HT_2CR-eYFP (yellow) constructs transiently transfected in HEK293 cells. The 5-HT-mediated increase in Ca_i²⁺ dye fluorescence over baseline was calculated as the % maximal response of WT 5-HT_2AR. (D) Representative BRET curve from HEK293 cells co-transfected with constant amount 5-HT_2AR-RLuc (100 ng; donor construct) and increasing amounts of the 5-HT_2CR-eYFP (1–20 fold excess; acceptor construct). (E) Representative BRET curve from HEK293 cells co-transfected with constant amount β₂-AR-Rluc (100 ng; donor construct) and increasing amounts of the 5-HT_2CR-eYFP (1–20 fold excess; acceptor construct). The mBRET signal was determined by calculating the ratio of light emitted at 528 nm to 460 nm (X 1000). Results obtained from four independent experiments.