Fig. 8.

DAT internalizes to the endosomal recycling compartment in response to phorbol ester treatment. DAT-PC12 cells were biotinylated at 4°C, warmed to 37°C, and treated with either vehicle (filled circles) or 1.0 μmβPMA (open squares) for 30 min at 37°C to initiate endocytosis. Cells were rapidly chilled to 4°C, and noninternalized biotin was released from the cell surface by treatment with 10 mm MesNa as described in Materials and Methods. Cells were homogenized and fractionated in 10–50% sucrose equilibrium gradients, and organelles were solubilized. Biotinylated proteins were isolated from each fraction with streptavidin beads and analyzed by 10% SDS-PAGE and immunoblot. Top, Immunoblot of internalized DAT and TfR after treatment with vehicle or 1.0 μmβPMA. Bottom, Quantitation of DAT and TfR immunoreactivity after treatment with either vehicle (filled circles) or 1.0 μm βPMA (open squares). A representative experiment of three independent experiments is shown.