FIGURE 4.

Detection of Ub∼thioesters in the HUWE1 HECT domains. A Ub-thioester assay with the indicated HUWE1 HECT domain proteins is shown. Purified HECT domains containing a C-terminal truncation of the last four amino acids were incubated with E1, UBE2L3, an ATP regenerating system, and Ub for the indicated amounts of time. Reactions were stopped with 4 m urea and non-reducing sample buffer (upper panel) or reducing sample buffer (lower panel), separated by SDS-PAGE, and analyzed by immunoblot with anti-Ub antibody.