Subcellular distribution of p53 in CLL cells. Cells from patient 21 were incubated in the presence of 100 μM ZVAD with no further additions (NA), 50 μM chlorambucil (chl) or 10 μM fludarabine (flu). Following 18-hour incubation, cells were fractionated by DDF as described in “Methods,” except that the pellet remaining following extraction with DDF buffer 1 was washed once with buffer 1 prior to extraction with buffer 2. The resulting fractions (Fn1, wash, Fn2 and Fn3) were analyzed by Western blotting. To allow direct visual assessment of p53 distribution, a volume of each fraction equivalent to 20 × 10⁶ cells was loaded on each lane. This resulted in the loading of 50 μg Fn1, 2 μg wash fraction, 20 μg Fn2 and 18 μg Fn3. LDH V, lactate dehydrogenase V; Hsp60, heat shock protein 60; COX IV, cytochrome oxidase IV; H2A, histone 2A. The p53 antibody detected a major band of 53 kDa and a minor band of 48 kDa, indicated by the asterisk. This band may correspond to the alternatively spliced form, p53β, described by Bourdon et al.⁴⁰  In this and all figures, lanes that were contiguous on autoradiograms are boxed together.