Biochemical characterization of a novel inhibitor of Rpn10, SB. (A) AlphaAssay screening for Rpn10 inhibitor (binder). Human recombinant Rpn10-GST and biotinylated Ub_2-7 were used. (B) Percentage of inhibition of Rpn10-Ub_2-7 binding by the 10 000 compounds screened. Internal figure: chemical structure of Rpn10 inhibitor (binder) candidate SB. (C) Dose response AlphaScreen assay of SB binding to Rpn10 or GST protein. (D) Recombinant human Rpn10 protein was incubated with control, SB (10μM), or NSC697923 (10μM) for 30 minutes at real time, then Ub_2-7 was added and incubated for 1 hour at real time. The mixture was then immunoprecipitated with Rpn10 antibody and subjected to immunoblotting using antibodies against Rpn10 and poly-Ub. (E) Measurement of K_d of hRpn10 with SB by FEB assay. Ten micrometers of compound or DiUb was applied on a recombinant His-hRpn10–immobilized graphene chip separately. DiUb and compound UNC0638 were used as positive and negative controls, respectively. The K_d is calculated as a median average over the test points. Three independent experiments were performed. Real-time changes of the I-response are shown in circles and correspond to the 1:1 binding model shown as solid lines. (F) Measurement of K_d of hRpn10 with SB by MST assay. K_d was derived from the binding response as a function of the GFP-hRpn10 concentration. Error in K_d represents fitting errors. (G) After being cultured with Dox for 3 days, AMO1 sgRNA-CT cells, AMO1 Rpn10-iKO cells, and iKO with Rpn10 adding back cells were treated with SB at different concentrations for 24 hours, followed by the cell viability being measured by a CTG assay (mean ± SD, n = 3).