FIGURE 6.

Ubiquitination activity of the HUWE1 HECT domains at 37 °C. a, the autoubiquitination activity of HUWE1 HECT domain at 37 °C was tested using 60 μm Ub as substrate and 2 μm WT HECT domains incubated with UBE1, UBE2L3, and an ATP regenerating system (note the different time scale for the two variants of the HECT domain). Reaction mixtures were separated on SDS-PAGE and analyzed by anti-Ub immunoblot. b, the Mcl-1 ubiquitination activity of HUWE1 HECT domain at 37 °C was tested using 5 μm FLAG-Mcl-1 as substrate and 100 nm WT HECT domains incubated with UBE1, UBE2L3, Ub, and an ATP regenerating system. Reaction mixtures were separated on SDS-PAGE and analyzed by anti-FLAG immunoblot.