35S-methionine-labeled PSMB4, CCT4, and GFP were translated in the PURE system with or without N-terminal fusions of either ubiquitin (Ub) or ubiquitin containing a mutated hydrophobic patch [Ub(3A)]. The G76V mutation in ubiquitin was used in the fusion. Soluble substrates (seeFigure S8A) were then incubated with E1, E2 (UBE2A), His-Ub, ATP, and recombinant UBR4 and KCMF1. The samples were then analyzed by autoradiography either directly (input) or following a His-Ub PD under denaturing conditions.
