FIGURE 1.

NT induces D2S internalization in transfected HEK-293 cells. HEK-293 cells transfected with D2S and NTR1 were exposed to receptor agonists (10 μm Quin and/or 10 nm NT) for 10 min to measure membrane-labeled D2R internalization and colocalization with Rab5 by immunofluorescence. A, immunolabeling against the FLAG epitope of membrane D2S-FLAG using two different secondary antibodies before and after permeabilization, such that the internalized signal appears in pure green, and the membrane signal appears in red and yellow. Scale bar, 15 μm. B, quantification of D2S internalization treatment measured by immunofluorescence and normalized against the total signal of each cell (n = 26 for control (Ctl), 29 for Quin, 37 for NT, and 36 for Quin+NT; results from three separate experiments). C, immunolabeling against the FLAG epitope of membrane D2S-FLAG (red) and GFP from cotransfected endosomal protein Rab5-GFP (green). Scale bar, 10 μm. D, colocalization of D2S and Rab5 signals analyzed by the Pearson correlation coefficient following treatment (n = 20 for every group). *, p < 0.05; **, p < 0.01; ***, p < 0.001, one-way ANOVA with Tukey's multiple comparison tests.