Luciferase complementation analysis indicates 5-HT_2AR:5-HT_2CR interaction within 50 nm.

(A) Schematic of luciferase complementation assay (LCA) between 5-HT2AR-CLuc and 5-HT2CR-NLuc constructs (adapted from Luker et al., 2004) [33, 34]. (B) Representative concentration response curve for 5-HT-mediated intracellular calcium (Cai2+) release for WT 5-HT2AR (red) and 5-HT2AR-CLuc (grey) constructs transiently transfected in HEK293 cells. The 5-HT-mediated increase in Cai2+ dye fluorescence over baseline was calculated as the % maximal response WT 5-HT2AR. (C) Representative 5-HT concentration response curve for WT 5-HT2CR (blue) and 5-HT2CR-NLuc (orange) constructs transiently transfected in HEK293 cells. The 5-HT-mediated increase in Cai2+ dye fluorescence over baseline was calculated as the % maximal response of WT 5-HT2CR. (D) Representative luminescence trace in HEK293 cells using varying transfection ratios (1:1, 2:1, 2:1, 3:1; 1 μg total plasmid DNA) of 5-HT2CR-NLuc and 5-HT2AR-CLuc constructs, respectively. Light counts/sec are shown at 1:2 (green), 1:1 (blue), 2:1 (red), and 3:1 (black) transfection ratios of 5-HT2CR-NLuc to 5-HT2AR-CLuc, respectively. (E) The maximal luminescence value from three independent experiments in which varying 5-HT2CR-NLuc:5-HT2AR-CLuc transfection ratios were tested. *p vs. 1:1 5-HT2CR-NLuc:5-HT2AR-CLuc transfection ratio.