Fig. 1.

Stable DAT expression in DAT-PC12 cells.A, Immunoblot of total cell extracts (25 μg/lane) from PC12 cells alone (PC12) or stably transfected with either vector (V-PC12) or hDAT (DAT-PC12) cDNAs. Blots were probed with a rat monoclonal antibody to the N terminus of the human dopamine transporter. B, Saturation kinetics of DAT-mediated (i.e., desipramine-insensitive) [³H]DA uptake in V-PC12 (filled circles) versus DAT-PC12 (open squares) cells. Assays were performed using increasing concentrations of [³H]DA, and nonspecific uptake was defined in the presence of 50 nm desipramine (V-PC12) or 50 nmdesipramine/500 nm nomifensine (DAT-PC12) in parallel for each [³H]DA concentration point. A representative experiment of three performed is shown. C, Dose–response curves of DAT antagonist treatment on DAT-PC12 cells. Cells were treated for 30 min with increasing concentrations of either GBR12909 (filled circles), mazindol (filled triangles), nomifensine (open squares), or imipramine (open circles) before initiating transport with 50 nm[³H]DA. Representative experiments are shown. All transport experiments were performed in the presence of 50 nm DMI to eliminate contribution by the endogenous NE transporter, and nonspecific transport was defined in the presence of 50 nm DMI and 500 nm nomifensine.