Fig. 9.

After PMA treatment, DAT is localized to the ERC. Organelle immunoisolation from DAT-PC12 cells. After treatment with 1.0 μm PMA for 30 min at 37°C, DAT-PC12 cells were homogenized and fractionated in 10–50% sucrose equilibrium gradients. Peak DAT–TfR fractions were pooled, and intact organelles were immunoisolated using magnetic beads coated with either a negative control antibody (mouse IgG) or a monoclonal antibody directed against the human transferrin receptor (αTfR; H68.4). Bead eluents and supernatants were analyzed by 10% SDS-PAGE and were immunoblotted with antibodies directed against either DAT or TfR. After PMA treatment, the majority of DAT and the TfR are precipitated by antibodies to the TfR, while both proteins remain in the supernatant when control beads are used.