FIGURE 5.
Binding of UBE3C to the proteasome is not sufficient for its activity. A, HeLa cell lines expressing the indicated exogenous UBE3C construct were immunoprecipitated (IP) with antibody against proteasome subunit PSMD2 using protein A/G-agarose beads. Input and bead-bound fractions were then analyzed by immunoblot (IB) against UBE3C. B, schematics of UBE3C constructs. UBE3C was expressed in cells with UBE3CNterm intact, removed, or replaced with the UBL domain of hHR23A. C, DD-GFP HeLa cells were stably transduced with no exogenous UBE3C or with the constructs shown in A. Cells were treated with siUBE3C or NTsi in the presence of stabilizing ligand and analyzed by flow cytometry at 3 h after ligand removal. DD-GFP cells expressing no exogenous UBE3C were included as control. Error bars show S.E.