(A) Relative expression of PSMB8 and PSMB9 in HEK293 cells upon 100 ng/ml IFNγ stimulation for 48 h. (B) PSMB8 or PSMB9 was immunoprecipitated for 48 h in 100 ng/ml IFNγ-stimulated HEK293 cells transiently overexpressing either PSMB8-FLAG or PSMB9-FLAG. Activity of PSMB8-FLAG or PSMB9-FLAG were assayed using 100 μM of either Ac-PAL-AMC for PSMB9 or Ac-ANW-AMC for PSMB8 in proteasome assay buffer with the indicated concentrations of TIR-199. Fluorescence signal upon release of AMC was measured in a Tecan plate reader. The results are presented as proteasome activity relative to the DMSO-treated control. Results are means ± S.D. for n=3 replicates. (C) The FLAG immunoprecipitates as in (B) were treated with either DMSO or 100 nM TIR-199 or 100 nM ONX-0914 for 30 min and activity assays were carried out with respective fluorogenic peptide substrates. **P<0.01; ns, not significant (compared with DMSO control treated, two-way ANOVA with Tukey’s multiple comparisons, mean ± SD from n=3 independent replicates). (D) TIR-199 (orange/blue) was docked into the crystal structure of human PSMB8 (cyan). Identified amino acid interacting residues are highlighted in black. Hydrogen bonds between TIR-199 and the amino acids in the binding pocket are shown by red dashed lines. Also refer to Supplementary Figure S2.