Bortezomib (Borte)-induced apoptosis and Bax activation in CLL cells. (A) Borte-induced apoptosis in 10 CLL samples. Fresh CLL cells were treated with different concentrations of Borte for 16 hours. Apoptotic cells were determined by the annexin V assay. (B) Borte-induced Bax protein accumulation in 9 cases of CLL patients. (C) Borte-induced Bax protein accumulation, oligomerization, and activation in 1 case of CLL patient. CLL cells were treated with Borte for 16 hours. Accumulation of Bax protein was determined by Western blotting using the anti-Bax antibody clone 2D2 at 1:1000 dilution. A total of 30 μg protein was loaded into each lane. Bax dimers and trimers are shown in the top panel blot indicated by 2× and 3×. For detection of conformationally changed Bax, the active form of Bax was immunoprecipitated (IP) by Bax 6A7 antibody and probed by Bax 2D2 antibody. (D) Bax translocation to mitochondria. CLL cells were treated with or without 20 nM Borte for 16 hours. Cells were stained with 100 nM MitoTracker for 15 minutes, washed 3 times, then fixed and permeabilized. Slides were stained with anti-Bax clone 3 antibody (1:20 dilution) for 1 hour and then stained with FITC-conjugated anti–mouse IgG (1:50 dilution) for 1 hour. Finally, slides were stained with 50 ng/mL DAPI. The red color indicates the mitochondrial location, and the yellow color represents the active Bax in the mitochondria.