Selective induction of proteasome β5-subunits in bortezomib-resistant cells. (A) Protein expression of proteasome β5-, β2-, and β1-subunits and α7-subunits in wild-type and bortezomib-resistant THP1 sublines. THP1/BTZ_(-100) represents a subline of THP1/BTZ₁₀₀ that was grown in the absence of bor-tezomib for 6 months. Note: 2 different sources of β5 antibodies were used: Biomol (PW8895; Plymouth Meeting, PA) and 20S X from Novus Biologicals (Littleton, CO), the latter indicated by an asterisk (*). (B) Induction of proteasome subunits β5, β2, and β1 in relation to resistance factors to bortezomib for the selected panel of bortezomib-resistant THP1 cells. Densitometry results are presented as the mean (± SD) of 4 separate experiments. (C) Native gel electrophoresis of crude cell extracts of THP1/WT cells and THP1/BTZ₁₀₀ cells subsequently analyzed for β5- and α7-subunit expression (left panels) and catalytic activity for the substrate Suc-LLVY-AMC (right panel). (D) Gel filtration of crude extracts of THP1/WT cells and THP1/BTZ₁₀₀ cells via a high-performance liquid chromatography (HPLC)–linked Superdex 200 HR 10/30 column (Supelco, Bellefonte, CA). Proteins were eluted by washing the column with 20 mM Tris-HCl, pH 7.5, 5 mM ATP, and 120 mM NaCl at a flow rate of 0.4 mL/min. Fractions were collected every minute and subject to Western blot analysis for β5 and α7. The column was calibrated with a mixture of purified proteins in the MW range of 16.6 kDa to 669 kDa.