FIGURE 3.

Monensin and leupeptin affect DAT trafficking, and TacDAT co-localizes with late endosomal marker Rab7 in HEK293 cells. A, intracellular accumulation measured by ELISA (described in Fig. 2D) in HEK293 cells transfected with Tac or TacDAT. The protease inhibitor leupeptin (leu) (100 μg/ml) or the recycling inhibitor monensin (mon) (25 μm) was included as indicated during the 1 h of internalization (means ± S.E. of n = 4; *, p < 0.05; **, p < 0.01; one-way ANOVA, Dunnett's multiple comparison test). B, surface expression determined in a surface ELISA of TacDAT, HA-DAT, control β₂-adrenergic receptor (β₂AR), isoproterenol (iso)-internalized β₂-adrenergic receptor, and EGFP-transferrin receptor (TfR) after 1-h treatment with monensin (25 μm) (means ± S.E. of n = 3–4; *, p < 0.05; **, p < 0.01; paired t test). C, confocal microscopy images of co-localization between TacDAT and EGFP-tagged Rab4, Rab7, and Rab11 after 1 h of Alexa Fluor 568-conjugated M1 antibody internalization in HEK293 cells. Left panels show Alexa Fluor 568 signal (M1), middle panels show EGFP signal, and right panels show the overlay of the two channels. Data are representative of at least three independent experiments.