Fig 1: Activation of PTHR1 signaling by abaloparatide and teriparatide treatment: (A) Activation of intracellular cAMP production: MC3T3‐E1 cells were challenged with 0–100 nmol/L of abaloparatide or teriparatide for 40 min at 37°C in the presence of IBMX. The medium was disposed and cells were snap frozen on liquid N3 before storage at 80°C. Intracellular cAMP was extracted and measured using ELISA assay as described by the manufacturer protocol. Each dose treatment was performed in duplicate and the experiment was repeated three times. The EC50 was calculated using results from three independent experiments. Results are the means ± SD. Upper panel: Absolute values are expressed as pmol/well. Lower panel: Values are expressed as % stimulation/maximal response. (B) Activation of β‐arrestin recruitment: PathHunter eXpress PTHR1 CHO‐K1 β‐arrestin GPCR assay was described under the material and methods section. CHO‐K1 cells were treated with 0–100 nmol/L of teriparatide or abaloparatide for 60 min at 37°C. Teriparatide and abaloparatide stimulations of β‐arrestin recruitment were determined by measuring light generation after adding β‐gal enzyme substrate. The EC50 was calculated using results from three independent experiments. Results are the means ± SD. Upper panel: Absolute values are expressed as relative light units (RLU). Lower panel: Values are expressed as % stimulation/maximal response. (C) Stimulation of PTHR1 internalization: PathHunter eXpress PTHR1 U2OS activated GPCR internalization assay was described under the material and methods section. U2OS cells were treated with 0–100 nmol/L of teriparatide or abaloparatide for 60 min at 37°C. PTHR1 internalization was determined by measuring light generation after adding β‐gal enzyme substrate. The EC50 was calculated using results from three independent experiments. Results are the means ± SD. Upper panel: Absolute values are expressed as RLU. Lower panel: Values are expressed as % stimulation/maximal response.