Figure 4: Sensitivity to oxidative inhibition is associated with DUB activation.

(a) Purified DUBs used in the assays. A known amount of BSA was used to estimate the concentration of the purified DUBs. (b) USP19 is highly sensitive to inhibition by H₂O₂. Purified USP19 or USP19CD was treated with the indicated concentrations of H₂O₂ at pH 7.4. The samples were diluted tenfold before the DUB activities were determined by the Ub-AFC assay. (c,d) The sensitivity of USP7 and UCH-L1 to inhibition by H₂O₂ is enhanced by high pH. USP7 or UCH-L1 was treated with H₂O₂ at the indicated pH and the DUB activity was determined by the Ub-AFC assay. (e) Ubiquitin activates USP7. USP7 was incubated at pH 7.4 in the absence or presence of ubiquitin (50 μM). The samples were diluted tenfold before Ub-AFC was added to determine the USP7 activities. (f) USP7 activation by ubiquitin sensitizes it to inhibition by H₂O₂. USP7 was pretreated in a buffer without or with ubiquitin for 5 min. Where indicated, H₂O₂ was also included. The USP7 activities were determined by the Ub-AFC assay at pH 7.4. The error bars indicate the mean of two independent experiments.