IPSI-001 inhibits the immunoproteasome. (A) Chemical structure of IPSI-001. (B) Competitive binding experiment to determine subunit binding profile of IPSI-001 in purified proteasomes from bovine pituitaries (20S) and spleen (20S_i). Purified proteasomes were incubated with 10 or 50 μM IPSI-001 or vehicle (Veh) for 20 minutes, and then [¹⁴C]-3,4-dichloroisocoumarin was added for an additional 30 minutes. Proteasomes were then separated by denaturing gel electrophoresis and the binding profile was identified using autoradiography. Subunit identifications were made based on the known electrophoretic migration patterns of immunoproteasome and constitutive proteasome subunits. (C) Competitive binding experiment to determine the subunit binding profile of IPSI-001 in ANBL-6 cells. Cells were treated for 4 hours with IPSI-001, followed by addition of the VS-L₃-AHx₃-dansyl reagent that binds to all unoccupied catalytic subunits. Protein extracts were then separated by denaturing gel electrophoresis and probed by Western blotting using an antidansyl antibody to visualize binding patterns. Antiactin was used as a loading control and anti-β1_i was used to confirm the relative position of β1_i. (D) In vitro assay for the 3 major catalytic activities of the proteasome in ANBL-6 cell lysates (5 μg) exposed to increasing concentrations of IPSI-001 in triplicate. Representative data are shown as the mean plus or minus standard deviation (SD) from 3 independent experiments. *P < .05 for proteasome inhibitory activity over control. (E) Western blot analysis of the accumulation of polyubiquitinated proteasome substrates in response to 50 μM IPSI-001 treatment in ANBL-6 cells over a period of 24 hours.