The synthesis of human ubiquitin and UbL probes has been described [24, 28] . IVT products were incubated with saturating amounts of the individual probes (0.2-0.4 mg/10 ml IVT lysate). Preincubation with NEM was performed for 10 min at room temperature at a final concentration of 10 mM, after RNase B treatment. Autoradiograms were subjected to quantification of the optical density with NIH Image software (version 1.32j) as ratio of labeled versus unlabeled IVT products. Purified human 26S proteasomes for the experiments in Figure 4 were purchased from Biomol International and inhibited with MG132 (50 mM). E. coliexpressed human recombinant USP14 for the experiments shown in Figure 3C 