Proteasome β5-, β2-, and β1-subunit mRNA levels and proteasome subunit–related catalytic activity in wild-type and bortezomib-resistant cells. (A) mRNA levels for proteasome β5-, β2-, and β1-subunits in selected variants of bortezomib-resistant THP1 cells relative to THP1/WT cells. mRNA levels were quantified using β-glucuronidase (GUS) as reference gene and depicted relative to THP1/WT cells. (B) Chymotrypsin-like, caspase-like, and trypsin-like proteasomal activities assayed with specific fluorogenic peptide substrates in cell extracts of THP1/WT, THP1/BTZ₃₀, THP1/BTZ₁₀₀, and THP1/BTZ₂₀₀ cells after a 4-day drug washout period (control) and after 24-hour incubation with 10 nM bor-tezomib (for THP/WT) and selective concentrations of 30 nM, 100 nM, and 200 nM bortezomib for the indicated bortezomib-resistant THP1 sublines. Controls for selective inhibition of caspase-like activity and trypsin-like activity included Ac-APnLD (25 μM) and leupeptin (20 μM), respectively. All results represent the mean (± SD) of 3 independent experiments. (C) Activity labeling of constitutive and immunoproteasome β-subunits in intact THP1/WT and selected bortezomib-resistant THP/BTZ cells using bodipyFL-Ahx₃L₃VS affinity probe.