Fig. 6.

Membrane raft-associated DATs do not undergo PMA-induced endocytosis. rDAT-LLCPK₁ cells were treated with vehicle or 200 nM PMA for 30 min at 37°C followed by cell surface protein biotinylation at 4°C. Cells were solubilized with 0.1% Triton X-100 followed by sucrose gradient centrifugation. (a) Aliquots of gradient fractions 5, 8, 9 and total homogenate (T) were incubated with NeutrAvidin beads and eluates of bound samples were immunoblotted for DAT. (b) Histogram indicating intensities of biotinylated bands from each fraction (means ± SE of three independent experiments). **, p < 0.01 relative to the corresponding vehicle treated control (Student’s t-test). (c) Immunoblot of PP1α performed on aliquots of biotinylated and non-biotinylated fractions of total homogenates.