cAMP measurement was performed in NG108-15 cells transiently expressing 5-HT6R using the BRET sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc) (41). NG108-15 cells were cotransfected in suspension with the 5-HT6R and CAMYEL constructs using Lipofectamine 2000 according to the manufacturer’s protocol and plated in white 96-well plates (Greiner) at a density of 80,000 cells per well. Twenty-four hours after transfection, the cells were washed with PBS containing calcium chloride and magnesium chloride (Gibco, ref 14040-091). Coelenterazine h was added at a final concentration of 5 μM. Plates were incubated at room temperature for 5 min before BRET measurements. BRET was measured using a Mithras LB 940 plate reader. Expression of 5-HT6R in NG108-15 cells induced a decrease in the CAMYEL BRET signal compared to that in cells transfected with an empty vector. This decrease in CAMYEL BRET signal was used as an index of 5-HT6R constitutive activity through Gs signaling.

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