b-AP15 targets USP14 and UCHL5 in MM cells. (A) MM.1S cells were treated with dimethylsulfoxide (DMSO) or b-AP15 (100 nM) for indicated times or treated with bortezomib for 3 hours; protein lysates were subjected to immunoblotting with anti-polyubiquitin or anti-actin antibodies. (B) MM.1S cells were treated with DMSO or b-AP15 for 3 hours; cells were lysed in lysis buffer and total protein was labeled with HA-Ub-VS probe and then subjected to immunoblotting with USP14 and UCHL5 antibodies. (C) MM.1S cells were treated with DMSO, b-AP15, or bortezomib for 3 hours; protein lysates were analyzed for proteasome activity. The percentage of proteasome activity was normalized to DMSO control (mean ± standard deviation [SD], n = 3). (D) Recombinant human HtrA2 enzyme was incubated with its substrate β-casein in assay buffer, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, silver staining, and quantification of cleaved β-casein. The bar graph represents the percentage of HtrA2-induced β-casein cleavage in the presence of bortezomib (3 nmol/L) or b-AP15 (100 nmol/L) vs DMSO control. Data represent means ± SD (n = 2; P < .05 for bortezomib). CT-L, chymotrypsin-like; T-L, trypsin-like; C-L, caspase-like.