Intracellular redistribution of cyt c in NB4 cells following treatment with CD437. (A) NB4 cells (300,000/mL) were treated for the indicated amount of time with vehicle (DMSO, Control) and with 10⁻⁶ mol/L CD437. Following harvesting and homogenization, a small aliquot of the homogenate was directly processed for Western blot analysis (total) and for the determination of DEVD-amc hydrolytic activity. The remaining amount of homogenate was subjected to differential ultracentrifugation to separate the mitochondrial fraction (Mit.) from the cytosol before Western blot analysis. Aliquots of the various fractions containing an equivalent amount of protein (30 μg) were subjected to Western blot analysis with antibodies specific for cyt c and actin. The molecular weight of protein standards is indicated on the right. The values of DEVD-amc hydrolytic activity are the mean ± SD of three replicates of the same homogenate. (B) NB4 cells (300,000/mL) were treated for 6 hours with vehicle (DMSO, Control) and with 10⁻⁶ mol/L CD437 following 2 hours preincubation in the absence or in the presence of z-VAD (100 μmol/L). Aliquots of the NB4 homogenates (total) or the cytosol fraction as in (A) containing an equivalent amount of protein were subjected to Western blot analysis with antibodies specific for cyt c and for actin. The molecular weight of protein standards is indicated on the right. The experiments are representative of at least four other experiments giving essentially the same results.