Dose-dependent effect of bortezomib on activity of the wild-type and N803A mutant HIF-1α CAD in various cell lines. The Gal4⁻ HIF-1α CAD constructs were cotransfected with a Gal4 reporter plasmid pFR-Luc (Stratagene, La Jolla, CA) using Effectene transfection reagent (QIAGEN, Valencia, CA). After exposure to the transfection mixture for 16 hours, the cells were trypsinized, replated, allowed to adhere for 5 hours, pretreated with bortezomib for 30 minutes, and exposed to normoxia or 0.5% O₂ hypoxia (in the presence of bortezomib) for 24 hours. Activities are expressed as the ratio of luciferase activity/protein concentration in arbitrary units (AU) and each of the bars represents the mean value (± SD) from 3 independent experiments.