Fig. 1. Neither bortezomib nor carfilzomib inhibits recombinant HtrA2/Omi in vitro (A) gel-based enzyme assay of recombinant human HtrA2/Omi protease using β-casein as a model substrate. Recombinant human HtrA2/Omi was pre-incubated with 100 μM ucf-101 (Lane 4), or bortezomib (BTZ) up to 100 μM (Lanes 5–15), each dissolved in DMSO, for 45 minutes at room temperature before 1 μg mL^–1 β-casein was added. After incubation at 45 °C for 1 hour, the reaction components were separated on a denaturing SDS gel and the separated proteins were visualized by Coomassie Brilliant Blue staining. (B) Recombinant human HtrA2/Omi was pre-incubated with the BTZ, carfilzomib (CFZ), or ucf-101, and analyzed in a gel-based enzyme assay using β-casein as a model substrate. (C) Peptide cleavage was assayed by using the fluorescent optimal substrate Mca-IRRVSYSF (K-Dnp) K. In the assays 10 μM fluorescent substrate was incubated with 30 pM human recombinant HtrA2/Omi for 60 minutes in the presence of BTZ, CFZ, or ucf-101. The solid line indicates a dose response curve for ucf-101 plotted against the % inhibition of HtrA2/Omi; dose response curves for either BTZ or CFZ could not be determined.