Figure 1. Screening of HECT E3 ligases to identify enzymes that assemble Lys³³-linked polyUb.

(A) Ubiquitylation assays of HECT E3 ligases in the presence of UBE1, Ub and different E2 enzymes: UBE2D1, UBE2D2, UBE2D3 and UBE2L3. (B) Ubiquitylated products generated by HECT E3 ligases with the optimal E2 were analysed by pRM LC–MS/MS for the abundance of Ub linkages as described in ‘Materials and Methods’ (Supplementary Figure S1B). Each Ub linkage assembled by the HECT E3 ligases was plotted as a bar graph where the y-axes are summed ion current values for the relevant daughter ions of each precursor mass analysed (Supplementary Table S2). No signal was observed for Met¹ and Lys²⁷ linkages for any of the ligases tested.