Bortezomib modulates Bcl-2 family protein levels and interactions. Effect of the ROS scavenger NAC and z-VAD-fmk on Noxa modulation. (A) Granta-519 and Jeko cells were treated with 20 nM bortezomib for 18 hours and total protein extracts were analyzed by Western blotting using suitable antibodies. Viability was quantified by annexin V/PI staining. (B) Granta-519 and Jeko cells were treated with 20 nM bortezomib for 16 hours, and Mcl-1 and Bak immunoprecipitations were performed as described in “Patients, materials, and methods.” Total extracts, and bound and unbound fractions were analyzed by Western blotting for Mcl-1 and Noxa or Bak and Mcl-1, respectively. ^*Unspecific band of immunoglobulin light chain from Bak antibody. (C) Granta-519 and Jeko cells were treated with 20 nM bortezomib for 20 hours in the presence or absence of 10 mM NAC or 50 μM z-VAD-fmk (both added 1 hour before bortezomib), and total protein extracts were analyzed for Noxa expression by Western blotting. The percentage of ROS and the loss of ΔΨ_m were determined by DHE and DiOC₆(3) staining, respectively, followed by fluorescence-activated cell sorter (FACs) analysis.