Effect of Hsp70 on CAD-induced chromatin DNA fragmentation in a cell-free system. (A) Homogeneity of Flag-Hsp70 protein purified from Flag-Hsp70–transfected 293T cells. Purified Hsp70 protein was electrophoresed in SDS-PAGE and stained with Coomassie brilliant blue. (B) Effect of purified Hsp70 on CAD-induced DNA fragmentation in a cell-free system. Caspase-3–activated CAD and various amounts (0-3 μL) of purified Hsp70 preparation were mixed. The volume of Hsp70 preparation was adjusted to become 3 μL by adding IgG immunoprecipitates (IgG ppt). The ability to induce chromatin DNA fragmentation was examined by electrophoresis in a 1.5% agarose gel (lanes 3-6). Chromatin DNA incubated without (lane 1) or with (lane 2) Hsp70 in the absence of CAD is shown. ICAD (0.3 μg) was incubated with CAD preparation to confirm that the DNA-fragmentation activity was dependent on CAD activity (lane 7). Percentages of DNA fragmentation calculated as in Figure 2 are shown at bottom.