Fig. 7. Activity of HECT E3 ligases and RBR E3 ligases under ByS reaction conditions. (A) The HECT domains of Nedd4-1, Nedd4-2, Itch and Wwp1 (2 μM) were incubated with either Ub(wt)–MES, Ub(K48R)–MES or Ub(K48R)–MES (200 μM) for 90 minutes at room temperature. The reaction mixtures were then analyzed using K63-ubiquitin linkage specific antibody. (B) GST-tagged RBR domains of Parkin (rat) and HHARI (human) (2 μM) were treated with Ub–MES as in (A) and were analyzed using K48-ubiquitin linkage antibody. HHARI and Parkin constructs lack auto-inhibitory domains.