Figure 6.

Nedd4–2 siRNA reduces PKC-dependent downregulation of untagged DAT. A, HeLa/DAT cells that were transfected twice with control (mock) or Nedd4–2 siRNA and used in experiments 6 d after the first transfection. The cells were incubated with vehicle or PMA as in Figure 2. The cells were then surface biotinylated and lysed, and the biotinylated proteins were pulled-down by NeutrAvidin agarose. Nonbiotinylated DAT was immunoprecipitated with rat anti-DAT from supernatants of NeutrAvidin agarose (B). DAT was detected by Western blotting using rat anti-DAT in NeutrAvidin precipitates (A) and DAT immunoprecipitates (B), whereas Nedd4–2 was probed in aliquots of lysates (C). The lysates were also blotted with antibodies to an unrelated protein (PP1) to control for sample loading (C). Quantification of the amount of biotinylated DAT was performed by densitometry (D). The data represent averaged values from five experiments (±SD) and are expressed as percentage of the amount of biotinylated DAT in vehicle-treated, mock-transfected cells. **p < 0.05 compared with mock-transfected, vehicle-treated cells.