Fig. 6.

DAT and TfR cofractionate on sucrose equilibrium gradients. DAT-PC12 cells were homogenized and fractionated on 10–50% sucrose equilibrium gradients as described in Materials and Methods. Fractions were TCA-precipitated and analyzed by SDS-PAGE (10%) and immunoblot. A, Immunoblot probing with antibodies for the human DAT, TfR, rab5A, and EEA1. B, Quantitation of 10–50% sucrose equilibrium gradient probed for DAT (filled circles), TfR (open triangles), rab5A (filled triangles), and EEA1 (open squares). DAT and TfR colocalize at 29 ± 0.6% sucrose (n = 5), whereas rab5A and EAA1 fractionated at a significantly distinct density (unpaired Student’st test; p < 0.005) at a sucrose concentration of 22.4 ± 0.4% sucrose (n = 3). Blots were scanned, and band densities were determined using ImageQuant software as described in Materials and Methods. Experiments were performed three to six times with essentially identical results. A representative example is shown.