Author response image 1. BAM22 drives noticeably less KOR recycling than DynB, even though they are both sensitive to ECE2 inhibition.

(A) Recycling of HA-KOR to the cell surface after 30 min treatment with 100 nM Dyn B or BAM22 and washout for 0-120 min. Cell surface receptors were quantified by ELISA as described in Methods. Levels of cell surface receptors before agonist treatment were taken as 100% for each individual experiment. % recycled receptors were calculated by subtracting surface receptors at t = 0 (30 min internalization) from each recycling time point. (B) Cell surface receptors quantified after peptides were washed out for 120 min in media without or with 20 μm or the ECE2 inhibitor (S136492). The data represent mean ± SEM from three independent experiments carried out in triplicate.