Fig 2: 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 ß5c, ß1c and ß2c proteasome activity; the substrates Ac-PAL and Ac-ANW were used to assay ß1i and ß5i 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).

Fig 3: PCA plots of USP14 and FASN inhibitor-treated LNCaP cells. The score plots of principal component analysis (PCA) obtained from the metabolomic analysis of LNCaP-LN3 cells treated with FASN and USP14 inhibitors in (a) positive and (b) negative ionization modes. Green, IU1-treated group; Red, Fasnall-treated group; Blue, IU1- and Fasnall-treated group; Black, control group (with DMSO); Yellow, vehicle group (without DMSO).

Fig 4: Identification of SGTA interacting DUBs.A) Beads coupled to recombinant Thioredoxin-SGTA fusion proteins as indicated or Thioredoxin only (Control) were incubated with rabbit reticulocyte lysate for 16 hours at 4°C. The beads were washed as detailed in Materials and Methods. Twice the bead volume of SDS sample buffer was added, and samples were processed for SDS PAGE and Western blotting and probed with antibodies against Bag6, UBL4A and Hsp70 proteins using a fluorescence- based Odyssey ® Fc Imaging System (LICOR). B) Beads described in A, were incubated with HeLa lysate for 16 hours, washed and processed for Western blotting as detailed in A. Blots were probed with antibodies against USP5, USP14, USP9X and USP10 proteins using fluorescence- based detection on an Odyssey ® Fc Imaging System (LICOR).

Fig 5: USP14 slightly reduces the ubiquitination of FASN. (a) HEK293T cells were transfected with His-FASN alone or in combination with Flag-USP14. Interaction between His-FASN and Flag-USP14 was detected by immunoblotting after immunoprecipitation with anti-flag antibody. (b) LNCaP cells were transfected with HA-ubiquitin alone or in combination with Flag-USP14 or His-FASN. MG132 treated for 6 h in cells before the harvest. FASN ubiquitination was observed by immunoblotting after immunoprecipitation with anti-HA antibody. (c) LNCaP cells were transfected with HA-ubiquitin alone or in combination with Flag-USP14 or His-FASN. IU1 (10 µM) treated for 24 h and MG132 treated for 6 h in cells before the harvest. FASN ubiquitination was observed using a Ni-NTA-mediated pull-down assay. TCL; total cell lysates.