Physical interaction of Hsp70 and CAD. TAg-Jurkat cells (5 × 10⁷) that had been transfected with expression vectors for Flag-Hsp70, HA-CAD, and ICAD were stimulated without or with staurosporine (A) or anti-CD3/CD28 Abs (B), and Hsp70 or CAD was immunoprecipitated with anti-Flag Ab (lanes 3, 5) or anti-HA Ab (lanes 4, 6). Precipitates were immunoblotted with anti-Flag Ab or anti-HA Ab, or anti-ICAD Ab. As control for blotting, 5% of cell lysates were directly immunoblotted with Abs (lanes 1-2). (C) Pull-down analysis. Recombinant His-Hsp70 or GST was coupled to beads and incubated with CAD released from the purified CAD/ICAD complex. After washing, precipitates were analyzed by immunoblot with anti-CAD Ab. (D) TAg-Jurkat cells were transfected with either Flag-Hsp70, Flag-Hsc70, or Flag-Hsp90 expression vector, together with HA-CAD and ICAD expression vector, and then stimulated with anti-CD3/CD28 Abs. Hsp70, Hsc70, or Hsp90 was immunoprecipitated with anti-Flag Ab and the precipitates were immunoblotted with anti-Flag or anti-HA Ab.