Mg 262

Purified recombinant Ubp6 variants (120 nM) and ADP-proteasomes (Figs. 2e, 3g: ubp6Δ hul5Δ i.e., nominally wild-type; Fig. 3g: rpt1-RK ubp6Δ hul5Δ, 5 nM) were assayed in reaction buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl_2, 0.5 mM DTT) supplemented with 5 mM “ATP-free” ADP. Proteasome and metalloprotease inhibitors were also supplemented in the reaction, which includes 0.5 mM ATPγS (Santa Cruz), 1.5 μM PS-341 (ApexBio), 7.5 μM MG-262 (Apexbio), 100 μM Epoxomicin (ApexBio), and 10 mM 1,10-phenanthroline (o-PA, Sigma). Ubp6 and ADP-proteasomes were preincubated for 15 min at 30 °C. 200 nM ADP-HA-Ub_n-NCB1 was then added to the Ubp6-proteasome mixture to initiate the reaction. Samples were subsequently analyzed by SDS-PAGE and immunoblotted using the following antibodies: 1:1,000 HA-HRP (Sigma), 1:50,000 Ubp6³⁶ , 1:10,000 Rpn13 (this study). All in vitro deubiquitination assays have been independently repeated. Yeast strains used are listed in Supplementary Table 3, and antibody information can be found in Supplementary Table 6.

Recombinant human IL1B, TNF and IFNG (R&D Systems, Minneapolis, MN) were dissolved in PBS containing 0.1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO). Budesonide (AstraZeneca, Mölndal, Sweden), dexamethasone (Steraloids, Newport, RI), MG-132 (Millipore Sigma, St. Louis, MO), MG-262 (APExBIO, Houston, TX), PR-171 (Adooq BioScience, Irvine, CA), E-64 (APExBIO, Houston, TX), PS-1145, ML-120B (both Sigma-Aldrich) and Org34517 (gift from Chiesi Farmaceutici, Parma, Italy) were dissolved in dimethyl sulfoxide as stocks of 10 mM. Cycloheximide (Sigma-Aldrich) was dissolved in water at 10 mg/ml.