Interaction of Hsp40 with the Hsp70/CAD complex. (A) TAg-Jurkat cells (5 × 10⁷) that had been transfected with expression vectors for Flag-Hsp40, Hsp70, HA-CAD, and ICAD were unstimulated (–) or stimulated (+) with anti-CD3/CD28 Abs or staurosporine (STS), and CAD was immunoprecipitated with anti-HA Ab. Precipitates were immunoblotted with anti-Hsp70 Ab, anti-HA Ab, or anti-Flag Ab. As control for blotting, cell lysates were directly immunoblotted with Abs. (B) Effect of Hsp40 on Hsp70-augmented CAD activity in a cell-free system. Caspase-3–activated CAD and various amounts (0-3 μL) of purified Hsp70 or purified Hsp90 preparation were mixed. At lane 6, 3 μg GST-Hsp40 purified from bacteria was added to the reaction. Then chromatin DNA was added to the reaction and DNA fragmentation was examined as in Figure 4. Percentages of DNA fragmentation calculated as in Figure 2 are shown at the bottom.