Figure 4

Inhibition of proteasome deubiquitinases. (a) Examination of 20S proteasome inhibition by hit compounds. Cell lysates (25 μg) were exposed to 20 μM of each compound except for bortezomib (BZ; 50 nM) for 5 min in assay buffer followed by the addition of substrates. The substrates Suc-LLVY, Z-LLE and Ac-KQL were used to assay β5c, β1c and β2c proteasome activity; the substrates Ac-PAL and Ac-ANW were used to assay β1i and β5i immunoproteasome activity. (b)19S proteasome preparations (10 nM) were incubated with ubiquitin rhodamine in the presence of the indicated compounds at 37 °C and fluorescence recorded; (c) 19S proteasome preparations (10 nM) were exposed to different compounds at 50 μM followed by labeling with HA-ubiquitin-vinylsulphone (HA-UbVS). The blots were probed with antibodies to HA, USP14 and UCHL5. Note the preferential loss of USP14 HA-UbVS labeling. The loss of recognition of USP14 following exposure to CB826 was observed in repeated experiments and remains unexplained (this phenomenon was not observed using extracts (see below)). The previously described DUB inhibitor b-AP15²⁸ was included as a reference. (d) Total cellular lysates from OPM-2 cells were incubated with ubiquitin rhodamine in the presence of 20 μM of the indicated compounds at 37 °C and fluorescence recorded. (e) OPM-2 cell extracts were incubated with compounds (20 μM) followed by HA-UbVS labeling. Filters were incubated with the indicated antibodies (also see Supplementary Fig. 3 where compounds were used at 50 μM).