TABLE 2

Trafficking of surface DAT in HEK-hDAT cells upon treatment for 1 h with dopamine (100 μM) or PMA (10 μM)

For the noncleavable biotin experiments, cells were pretreated for 1 h at 37°C with 100 μM dopamine or 10 μM PMA, drug was removed, and noncleavable biotinylation was applied. For the cleavable biotin method, cell surface protein was biotinylated before treatment for 1 h at 37°C with 100 μM dopamine or 10 μM PMA; biotin was stripped with 2-mercaptoethanesulfonic acid from non-endocytosed protein, and subsequently, the remaining biotinylated protein was isolated from cell extracts. Control incubation signifies incubation without dopamine or PMA for the same time as with dopamine or PMA. The glutathione strip of untreated cells assesses incomplete stripping, which would cause non-endocytosed DAT to show up as endocytosed DAT. Results are mean ± S.E.M. of three independent experiments.