NanoBiT β-Arrestin Recruitment Assay for PTH1R

β-arrestin recruitment to PTH1R was measured using the NanoBiT β-arrestin-recruitment assay^{52 (link)} with minor modifications. In brief, plasmid transfection was performed in a 6-cm culture dish with a mixture of 200 ng N-terminal large BiT-fused β-arrestin 1 (Lg-β-arrestin 1) or Lg-β-arrestin 2 and 1,000 ng C-terminal small BiT-fused PTH1R (PTH1R-Sm) plasmids. After 24 h of incubation, the transfected cells were collected using 0.53 mM EDTA-containing D-PBS, centrifuged at 190g for 5 min and suspended in 4 ml of the assay buffer described for the GloSensor assay. The cell suspension was dispensed into a white 96-well plate at a volume of 80 μl per well (hereafter 96-well plate) and loaded with 20 μl of 50 μM coelenterazine (Carbosynth) diluted in the assay buffer. After 2 h of incubation at room temperature, the plate was measured for baseline luminescence (SpectraMax L equipped with 2PMT, Molecular Devices; SoftMax Pro (v.7.03), Molecular Devices) and 20 μl of 6× ligand diluted in the assay buffer or vehicle was manually added. The plate was read for 15 min with a 40 s interval at room temperature. The luminescence counts from 13 to 15 min after ligand addition were averaged and normalized to the initial counts.  the response were fitted to all data using the same procedure as described for the GloSensor assay.