Fig 1: ISG20L2 competes with PSMB5 in BTZ binding.(A) Molecular structure of biotinylated BTZ (BTZ-b). (B) Flow cytometry–based apoptosis assay to examine the cytotoxicity of BTZ-b in CTR-KD versus ISG-KD (sh1) ARD cells. The cells were treated with BTZ-b (2 μM) for 24 hours. DMSO- or biotin-treated cells served as CTRs (n = 3). Student’s t test was performed. *P ≤ 0.05. (C) BTZ-b pull-down assay using ARD cell lysate in the presence of BTZ to examine BTZ competition with BTZ-b in PSMB5 binding. BTZ was added to the cell lysate to a final concentration of 200 μM and incubated with BTZ-b–coated beads for competition. (D) Biotinylated BTZ pull-down assay using CTR-KD and ISG-KD ARD cell lysates. Biotin served as a negative control for pull-down. The pull-down precipitate and the whole-cell lysates (input) were subjected to Western blotting. (E) BTZ-b pull-down assay using CTR and ISG-OE AMO1 cell lysates. (F) BTZ-b pull-down assay using recombinant ISG20L2 (rISG) and PSMB5 (rB5) proteins. The recombinant protein was dissolved in PBS. The final concentration of rB5 was 0.5 μg/mL (+), while the final concentration of rISG was 0.25 μg/mL (+) or 1.5 μg/mL (++). Using the gray intensity of PSMB5 pull-down by BTZ-b alone as a control, ImageJ software (NIH) was used to determine the levels of PSMB5 pull-down by BTZ-b with rISG competition. Numbers indicated on the lane. (G) BTZ binds to ISG20L2 as shown by SPR measurements. Graphs of equilibrium response unit (RU) responses versus compound concentrations were plotted. The estimated KD is 26.71 μM, as shown by the vertical dotted line. (H) Schematic mechanism of ISG20L2-induced MM BTZ resistance.