Figure 5

Time dependence of β-arrestin2 recruitment of μ-OR in ΔGRK cell lines. Recruitment of β-arrestin2 after 6 min (a,c,e) or 60 min (b,d,f) stimulation by saturating concentrations of DAMGO (37.5 µM), fentanyl [3.3 µM (a,b) or 10 µM (c–f)], loperamide [3.3 µM (a,b) or 10 µM (c–f)] or morphine (37.5 µM) measured by three different assays: ebBRET (a,b), recruitment of β-arrestin2-RlucII to μ-OR-EYFP (c,d), or recruitment of GFP²-β-arrestin2 to μ-OR-RlucII. Data represent the mean of individual experiments (circles) as well as the mean ± SEM (columns) after buffer subtraction and normalization to the maximum response in parental (WT) cells stimulated with DAMGO for 6 min from 3–4 independent experiments performed in duplicate. BRET ratios in knockout cells were compared with parental cells by repeated measures one-way ANOVA with Dunnett’s multiple comparisons test, except for ebBRET assay with 60 min loperamide stimulation, which was compared by mixed-effects analysis with Dunnett’s multiple comparisons test. *P = 0.01–0.05, **P = 0.001–0.01, ***P < 0.001.