PLA was used to determine the presence of GPCR-RAMP complexes in HEK293T cells cotransfected with a subset of GPCRs and RAMP2 that were chosen on the basis of SBA results. Gelatin-coated coverslips were placed within the wells of a six-well dish with one well per transfection condition. HEK293T cells were seeded at 100,000 cells/ml and allowed to grow for 24 hours before transfection. The cells were transfected with 0.4 μg (except in one experiment where 0.5 μg was used) of pcDNA3.1(+) vector encoding the GPCR and RAMP2 constructs (see DNA constructs in Materials and Methods for more information). The total amount of DNA used for each transfection was brought to 2 μg with an empty pcDNA3.1(+) vector. For the mock transfection, 2 μg of empty pcDNA3.1(+) vector was used. Transfection was performed with 4 μl of Lipofectamine 2000 per well of the six-well dish. The total volume of media per well was maintained at 2 ml. After 24 hours, cells were washed twice in PBS, fixed and permeabilized with ice-cold methanol for 5 min at −20°C, and then washed three times with PBS. For the FA fixation, cells were fixed for 10 min at RT with 4% weight:volume FA (Polysciences Inc.) in 1× PBS (final concentration). After fixation, cells were washed three times in PBS and then processed following the manufacturer’s instructions for DuoLink In Situ Detection Reagents Red Mouse/Rabbit (DUO92101) using rabbit anti-HA (Cell Signaling Technology) and mouse anti-FLAG (Sigma-Aldrich) primary Abs. After PLA processing, cells were mounted in DuoLink mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich), allowed to incubate at RT, stored overnight at −20°C, and imaged the following day.

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