Inhibition of proteasome activity by bortezomib and accumulation of proteasome-degraded proteins. (A) Granta-519 cells were incubated with bortezomib (10-50 nM) from 30 minutes to 3 hours, and proteasome activity was analyzed as described in “Patients, materials, and methods.” (B) Total protein extracts (50 μg) from Granta-519 cells were incubated in the absence (-) or presence (+) of 20 nM bortezomib for 6 hours and analyzed by Western blotting. Membranes were probed for p53, p21, and pIκB to confirm proteasome activity inhibition. α-tubulin was also probed as an equal loading control. (C) Primary MCL cells from patient no. 4 were incubated with bortezomib (20-100 nM) for 3 and 6 hours, and proteasome activity was analyzed as described in “Patients, materials, and methods.” (D) Cells from patient 4 were incubated with the indicated doses of bortezomib for 6 hours. Whole protein extracts were obtained, and p53, p21, and pIκB protein levels were analyzed by Western blotting. Data are given as the mean value ± SD of 2 independent experiments.