FIGURE 3.

WW3 and WW4 domains of Nedd4-2 are necessary for DAT ubiquitination. A, schematic representation of wild-type and mutant YFP-FH-Nedd4-2 constructs. B, HEK293T cells stably expressing FH-DAT (HEK293/FH-DAT) were transfected with empty vector (mock), wild-type, or mutant YFP-FH-Nedd4-2. After 2 days the cells were treated with 1 μm PMA for 15 min and lysed. DAT was immunoprecipitated using Nt-DAT antibody, and the immunoprecipitates were probed by Western blotting (WB) with ubiquitin (P4D1) and, subsequently, Nt-DAT antibodies. YFP-tagged Nedd4-2 constructs were detected in cell lysates using the Nedd4-2 antibody. C, quantification of several experiments performed identically to the experiment presented in B. Bars represent the mean values (±S.E.) of ubiquitinated DAT normalized to the amount of total DAT in arbitrary units (Ub/DAT). All Ub/DAT values are calculated as percent of the Ub/DAT value in mock-transfected cells. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 3). D, HEK/CFP-DAT cells were co-transfected with either non-targeting (NT) or duplex 4 siRNA (targeting Nedd4-2) and various DNA plasmids: wild-type YFP-FH-Nedd4-2, YFP-FH-Nedd4-2 resistant to siRNA duplex 4 (resistant form, rf), and YFP-FH-Nedd4-2-mWW1,2-rf or YFP-FH-Nedd4-2-mWW3,4-rf. After 2 days the cells were treated with 1 μm PMA for 15 min and lysed. DAT was immunoprecipitated using the Nt-DAT antibody, and the immunoprecipitates were probed with ubiquitin (P4D1) and, subsequently, Nt-DAT antibodies. YFP-tagged Nedd4-2 was detected in cell lysates using Nedd4-2 antibody. The amount of ubiquitinated DAT normalized to the amount of total DAT (Ub/DAT) is indicated for each experimental variant. The experiment is representative of two independent experiments with similar results.