AI-CRISPR and HAR-CRISPR βArr1/2 KO cells were maintained in MEM supplemented with 10% FBS and 1% penicillin/streptomycin or gentamicin (20 μg/ml). SL-CRISPR βArr1/2 KO cells were maintained in DMEM without pyruvate supplemented with 10% FBS and 1% penicillin/streptomycin or gentamicin (20 μg/ml). For ERK1/2 activation assays of cells transiently expressing β2AR, CRISPR βArr1/2 KO cells in 10-cm dishes at 40 to 50% confluence were transiently cotransfected with 2 μg of pcDNA3.1-FLAG-β2AR and 1 μg of plasmid encoding HA epitope–tagged βArr2 (pcDNA3.1-HA-βArr2) or 1 μg each of pcDNA3-HA-βArr2 and a plasmid encoding HA epitope–tagged βArr1 (pcDNA3.1-HA-βArr1) using Lipofectamine 2000 according to the manufacturer’s protocol. Equal amounts of empty vector were used for mock-transfected controls. Twenty-four hours after transfection, cells were plated onto six-well dishes. Stimulations were performed 16 to 24 hours later following 4 hours of serum deprivation. The cell surface abundance of β2AR was determined by [125I](−)-iodocyanopindolol binding. Western blotting analysis of whole-cell lysates with the A1CT anti-βArr1/2 antibody was used to assess the abundance of βArr2 or βArr1/2. For assays of cells transiently expressing β1AR, CRISPR βArr1/2 KO cells in 10-cm dishes were transiently cotransfected with 4 μg of pcDNA3-FLAG-β1AR and 50 ng of pcDNA3.1-HA-βArr1 and 250 ng of pcDNA3.1-HA-βArr2 using FuGene6 according to the manufacturer’s protocol. Equal amounts of empty vector were used for mock-transfected controls. Twenty-four hours after transfection, cells were split into six-well dishes and serum-deprived overnight before stimulation. β1AR cell surface abundance was assessed by FITC-conjugated anti-FLAG labeling, and βArr1/2 abundance was assessed by Western blotting analysis with the A1CT anti-βArr1/2 antibody. For assays of cells transiently expressing V2R, CRISPR βArr1/2 KO cells were seeded in 10-cm dishes at an initial density of 7.5 × 105 cells per dish. Twenty-four hours later, the cells were transfected with 3 μg of pcDNA3.1-HA-V2R with or without 200 ng of pcDNA3.1-FLAG-βArr1 or pcDNA3.1-FLAG-βArr2 (77) using the calcium phosphate DNA precipitation method as previously described (110). Equal amounts of empty vector were used for mock-transfected controls. The next day, cells were detached with phosphate-buffered saline (PBS) and 5 mM EDTA and plated onto poly-l-lysine–coated 12-well plates. Stimulations were performed 24 hours later following 30 min of serum deprivation. Western blotting analysis of whole-cell lysates with the A1CT anti-βArr1/2 antibody was used to assess the abundance of βArr2 or βArr1/2. For assays of cells transiently expressing FSHR, CRISPR βArr1/2 KO cells in 10-cm dishes at 30% confluence were cotransfected with 2 μg of pcDNA3-FLAG-FSHR and 200 ng each of pcDNA3.1-HA-βArr1 and pcDNA3.1-HA-βArr2 or 400 ng of empty vector using METAFECTENE transfection reagent according to the manufacturer’s protocol. Twenty-four hours after transfection, cells were split into 12-well dishes. After a further 24 hours, cells were serum-starved for 4 to 6 hours before stimulation. Plasma membrane FSHR abundance was assessed using Terbium-conjugated anti-FLAG antibody, whereas βArr1/2 abundance was assessed by Western blotting analysis of whole-cell lysates with the A1CT anti-βArr1/2 antibody.

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