Fig. 3.

Inhibition of OX1R activation by JH112, suvorexant and SB-674042. Inhibition of the orexin A effect was determined in (A) an IP₁ accumulation assay (Cisbio), (B) a Gα_q-RlucII/Gγ-GFP10 biosensor BRET assay, (C) a β-arrestin recruitment assay based on enzyme fragment complementation (DiscoverX), and (D) a BRET assay for the recruitment of β–arrestin-2 to the plasma membrane. In each case, orexin A concentration-response curves were collected in the presence of a given concentration of the antagonist. While SB-674042 mostly leads to a decrease of the orexin A potency (rightward shift of EC₅₀), JH112 and suvorexant additionally depress the maximum response (E_max) of orexin A, especially for the recruitment of β–arrestin-2. Curves represent the mean ± SEM from 3 to 16 individual experiments, each performed in duplicate (A and C) or triplicate (B and D).