USP14 reacts more efficiently with ISG15VS in its proteasome-associated form USP14 and its yeast counterpart Ubp6 show significantly higher activity when bound to the 26S proteasome [29] . This activity may in fact be strictly dependent on association with the proteasome, as shown for Ubp6 [30] . As further evidence for a physiological role of the interaction of USP14 with ISG15, we investigated whether the allosteric activation of USP14 also influences its reactivity toward ISG15VS. We examined labeling of USP14 with the ubiquitin-and the ISG15-based probes as a function of the concentration of added purified proteasomes. As a negative control, we evaluated USP5, a DUB that is not a known interaction partner of the 26S complex in vivo. As anticipated, the inclusion of purified proteasomes had no effect on the ISG15VS-or UbVME-reactivity of USP5 ( Figure 4B ). In contrast, we observed a dose-dependent increase in ISG15VS adduct formation of USP14 with increasing proteasome concentration, indicating enhanced activity of this DUB. The effect was similar in magnitude to that seen for the ubiquitin probe ( Figure 4A , C).