Cell culture and transfection
Human embryonic kidney (HEK293T) cells were maintained in a humidified incubator at 37°C with 5% CO2 and grown to 90-95% confluence in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U ml-1 penicillin and 100 µg ml-1streptomycin. Cells were transfected using Lipofectamine 2000 according to the manufacturer’s recommended protocol. All transfections were performed under serum-free conditions in Opti-MEM. Transfections were allowed to proceed for 4-5 h before the media was changed back to DMEM with 10 % FBS. Experiments were run 24 h after transfection. For cAMP assays, cells were plated in 6-mm dishes. DNA was kept constant at 6 µg and 6 µl of Lipofectamine2000 per plate was used. Empty vector (pcDNA3.1+) was used to adjust the total amount of DNA. The lack of cAMP stimulation in control vector (pcDNA3.1+) transfected cells and the robust change in cAMP levels in D1/NOP transfected cells after treatment with specific ligands were used to establish the success of transfection (Feng et al. , 2017). This is possible because HEK293T cells do not natively express D1 and NOP receptors.