Fluorescent ub

The E2 discharge assay to monitor ubiquitin (Ub) transfer from E2 to HUWE1 was performed in two steps. First, UbcH5B at 9.1 μM (R&D Systems) was reacted with UBE1 at 0.42 μM (R&D Systems) and fluorescent Ub at 16.7 μM (R&D Systems) buffered in 50 mM Tris 7.6, 300 mM NaCl. This reaction was initiated with 2 mM Mg-ATP (R&D Systems) for 30 min at room temperature and then quenched by diluting four-fold with 25 mM HEPES 7.5, 100 mM NaCl, 25 mM EDTA. The pre-charged E2-Ub mixture was mixed with excess wild-type or mutant HUWE1 at room temperature in a total reaction volume of 15 μl with 0.08 μM of E2-Ub and 0.4 μM HUWE1. At the indicated reaction times, the reactions were quenched with 4x SDS sample loading buffer lacking reducing agent. Reaction mixtures were separated by SDS-PAGE and gels were analyzed by fluorescent imaging on a Typhoon FLA 9500 (GE Life Sciences). Intensities of the bands were quantified by ImageJ and normalized to the E2-Ub only control. Error bars signify standard deviation and are generated from triplicate measurements. All plots were generated with prism 7.0.