Agonist-induced signaling and internalization of Rog. (A) Maximum calcium response plotted as a function of spiradoline dose for cells transfected with Rog or the wild-type KOR and treated with dynorphin or spiradoline. (B) Confocal micrographs show representative internalization of GFP-tagged Rog receptors 1 h after treatment with 10 μM or 100 μM spiradoline. Dynorphin at 100 μM (far right) caused little internalization in this assay. (C) ELISA for FLAG-tagged cell-surface receptors shows dose-dependent loss of receptors from cell surface one h after spiradoline treatment. After a 1 μM dose of spiradoline, internalization is evident within 15 minutes. Data are expressed as a percentage of receptors detected on surface of untreated cells expressing Rog. Error bars represent SEM for three replicates.