The quantification of the cell surface fluorescence with FACS after DOR agonist challenge: the effect of (1DMe)NPYF on the agonist- induced internalization of DOR. The cells were first treated with 100 nM DPDPE alone or 100 nM DPDPE + 100 nM (1DMe)NPYF or (1DMe)NPYF at 37°C for indicated times after which the cell surface receptors were detected with an anti-MYC-antibody and a fluorescent secondary antibody. 10000 cells/sample were analyzed with FACS. The combined data of five different experiments performed in duplicates is shown. The data is presented as the internalization percent that is calculated relative to the time-matched control cells (see Experimental section). The statistical significance was analyzed from the non-normalized raw data with two-way ANOVA (Bonferroni's post test, variance as SD, n = 4). *p < 0.05; **p < 0.01 and ***p < 0.001 shows statistical significance relative to the basal level (untreated time-matched cells), †††p < 0.001 significant change in cell surface fluorescence between treatments. The dashed bars represent cells treated with 100 nM DPDPE alone, the solid bars cells treated with 100 nM DPDPE + 100 nM (1DMe)NPYF and the dotted bars represent the cells with 100 nM (1DMe)NPYF alone.