FIGURE 5.

Recombinant Nedd4-1 catalyzes the mono-ubiquitination of hDCNL1. Purified Nedd4-1 (5–80 ng) was used for E3 titration (panels A and C) and time course (panel B) experiments in vitro with GST-hDCNL1 (panels A and B) or His-hDCNL1 (panel C), as described under “Experimental Procedures.” Production of mono-ubiquitinated products was quantified and is presented graphically. In panel D, GST-hDCNL1 (50 ng), in the forms of full-length (lanes 1 and 2) or truncations (amino acids 1–57, lanes 3 and 4; amino acids 58–259, lanes 5 and 6), was subject to the ubiquitination assay in the presence of or absence of Nedd4-1.