CHO cells (ATCC) were transiently transfected with wild-type or mutant β2-AR, and COS-1 cells (ATCC) were transiently transfected with wild-type or mutant α1B-AR following the manufacturer’s protocol (FuGENE HD, Roche). Assays measuring cAMP accumulation were performed 48 hours after transfection following the manufacturer’s instructions (LANCE Ultra cAMP kit, PerkinElmer). In brief, increasing concentrations of isoproterenol (100 pM to 100 μM) (β2-AR) or increasing concentrations of NE (100 pM to 100 μM) in the presence of a saturating concentration (10 μM) of propranolol to remove β-AR responses (α1B-AR) were added to 1000 transfected cells in stimulation buffer in a white 384-well plate (OptiPlate, PerkinElmer Life Sciences). The plates were incubated for 30 min at room temperature. Cells were then lysed by the addition of the europium (Eu) chelate–labeled cAMP tracer and the cAMP-specific monoclonal antibodies labeled with the ULight dye, diluted in cAMP detection buffer (LANCE Ultra cAMP kit, PerkinElmer), followed by incubation for 1 hour at room temperature. The emission signals were measured at 615 and 665 nm after excitation at 340 nm using a Tecan microplate reader (Tecan).

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