FIGURE 5.

UBE2D1-3 and UBE2L3 ubiquitin-conjugating enzymes are involved in DAT ubiquitination. A, HEK/CFP-HA-DAT cells were transfected with NT siRNA, Nedd4-2 siRNA, siRNA targeting UBE2D2-3, UBE2E3, UBE2L3, or a mixture of siRNAs targeting UBE2D2-3 and UBE2L3. Cells were treated with 1 μm PMA for 15 min, lysed, and DAT was precipitated with the HA11 antibody. Following electrophoresis and transfer, DAT immunoprecipitates (IP) were probed with antibodies against ubiquitin (P4D1) and, consecutively, with the monoclonal GFP antibody. Total cell lysates were blotted with antibodies to Nedd4-2, E2 enzymes, and actin (loading control). B, quantification of several siRNA experiments exemplified in A. Bars represent the mean values (±S.E., n = 3) of ubiquitinated DAT normalized to the amount of total DAT in arbitrary units (Ub/DAT). *, p < 0.05; **, p < 0.01. WB, Western blot.