Figure 4. The Nedd8 binding sequence is critical for the autoneddyaltion of Smurf1.

(A) Smurf1-HECT WT or 10 A mutant was co-expressed with Nedd8 in HEK293T cells and the expression of Smurf1 was analyzed by western blotting. (B) In vivo Smurf1 neddylationassay. HEK293T cells were transfected with Flag-Nedd8, Myc-tagged Smurf1 WT and mutants. The cells were harvested and subjected to neddylation assay 48 h after transfection. (C) In vitro autoneddylation assays were performed using purified wild-type Smurf1 or 10 A mutant together with Nedd8. Autoneddylated Smurf was analyzed by immunoblotting using anti-nedd8 antibody. (D) WT-Smurf1 HECT, Smurf1-HECT-10A mutant efficiently form Nedd8 thioester intermediates. Wild-type, 10 A mutant and C426A mutants were charged for 5 min at room temperature and analyzed by immunoblotting under non-reducing dithiothreitol (−DTT, lanes 1–3) or reducing conditions (+DTT, lanes 4–6) using the Nedd8 antibody.