β-arrestin2 recruitment assay in opioid receptor expressing βarr2eGFP-U2OS cells. The cells are live imaged, and the βarr2 recruitment evoked by drug induced receptor activation is visualized as bright green punctae (white arrows) by confocal microscopy. (A) MOR expressing cells are treated with DAMGO or NAQ (both 10 μM) as indicated. DAMGO induces robust βarr2 recruitment, while NAQ does not. In addition, NAQ pretreatment completely blocks DAMGO induced βarr2 recruitment. These findings are consistent with NAQ acting as an antagonist at the MOR. (B) DOR and KOR expressing cells are treated with a control agonist (SNC80 or U69,593, 10 μM, as indicated) or 100 μM NAQ. The control agonist induces robust βarr2 recruitment, while NAQ induces fewer, dimmer punctae than the control agonist. These findings are consistent with NAQ acting as a partial agonist at the DOR and KOR.