FIGURE 1.

PICK1 co-clusters with Tac DAT C24 but not with TacDAT. a, schematic representation of the fusion proteins Tac DAT C24 and TacDAT. TacDAT C24 is a fusion protein between Tac and the 24 C-terminal residues of DAT with an N-terminal M1 antibody FLAG epitope. TacDAT is a head-to-tail fusion of Tac (α-subunit of the IL2 receptor) (green) with an N-terminal M1 antibody FLAG epitope (DYKDDDDK) and full-length DAT (blue) (43). b, confocal laser scanning micrographs of Flp-In T-REx 293 eYFP-PICK1 cells induced with tetracycline and transiently expressing TacDAT C24 (top panel) or TacDAT (bottom panel). Cells were surface-labeled with Alexa Fluor 568-conjugated anti-FLAG antibody to visualize surface TacDAT C24 (top middle panel) and TacDAT (middle bottom panel) in transfected cells. The eYFP-PICK1 signal (green) is shown in the left panels and illustrates the lack of clustering and partial plasma membrane recruitment in TacDAT-transfected cells. In contrast, profound eYFP-PICK1 clustering was seen in TacDAT C24-transfected cells. Nuclei were highlighted in the top panel to demonstrate the juxtanuclear localization of the eYFP-PICK1 clusters. c, confocal laser scanning micrographs of Flp-In T-REx 293 eYFP-PICK1 cells induced with tetracycline and transiently expressing TacDAT. Cells were surface-labeled with Alexa Fluor 568-conjugated anti-FLAG antibody and internalized with PMA for 25 min (top middle panel). Subsequently, the cells were treated with staurosporine (Stau; 1 μm) for 60 min to allow potential recycling (bottom middle panel). The eYFP-PICK1 signal (green) is shown in the left panel and illustrates the lack of marked clustering in the TacDAT-internalized cells. Representative images are shown from ∼15 cells visualized per condition in each experiment and over three separate experiments. The small squares mark areas that are shown enlarged inside large squares. Scale bar, 15 μm.