FIGURE 5.

Chimeric receptor DOR DAT8 does not cluster eYFP-PICK1. a, schematic representation of DOR DAT8 showing the N-terminal FLAG tag sequence followed by the full-length DOR and the last 8 PICK1 binding C-terminal residues of DAT. b, confocal laser scanning micrographs of Flp-In T-REx 293 eYFP-PICK1 cells transiently expressing DOR DAT8. The pictures show agonist-induced internalization of DOR DAT8 (compared with untreated control) that was surface-labeled with Alexa Fluor 568-conjugated anti-FLAG M1 antibody at 4 °C without (−Tet) and with (+Tet) induced expression of eYFP-PICK1 (green). Internalization was stimulated by 10 μm δ-opioid receptor agonist peptide DADLE for 25 min at 37 °C followed by a 60-min incubation at 37 °C with 10 μm antagonist Naxo to allow potential recycling of receptors. Panels starting from the left show the DOR DAT8 signal in non-induced cells, DOR DAT8 signal in tetracycline-induced cells (red), eYFP-PICK1 signal (green), and merge of red and green signals. Small white squares mark areas that are shown enlarged inside large white squares. Pictures are representative of ≈15 cells visualized per condition in each experiment and over five separate experiments. Scale bars, 15 μm.