Figure 5.
Figure 5. Functional domains of Hsp70 to bind and activate CAD. (A) A scheme of wild-type Hsp70 and its deletion mutants. ATP-binding domain (1-385 amino acids), linker region (385-393 amino acids), and peptide-binding domain (393-640 amino acids) are indicated on the top. ATP-binding domain–deleted Hsp70 mutant (Hsp70ΔABD), peptide-binding domain–deleted Hsp70 mutant (Hsp70ΔPBD), and peptide-binding domain fragment (Hsp70PBD) are shown. (B) Association of CAD with Hsp70 and its mutants. Treated with 1 μM staurosporine were 293T cells that had been transfected with expression vectors for Flag-Hsp70 or its mutants (Hsp70ΔABD, Hsp70ΔPBD, or Hsp70PBD), together with expression vectors for HA-CAD and ICAD, and Hsp70 or its mutants were immunoprecipitated with anti-Flag Ab from cell lysates. Precipitates were immunoblotted with either anti-HA Ab or anti-Flag Ab. To estimate the expression of CAD protein in each cell lysate, an aliquot of each cell lysate was immunoblotted with anti-HA Ab. (C) Left panel: homogeneity of purified Hsp70 mutant proteins. Hsp70 or its mutant proteins (Hsp70ΔABD, Hsp70ΔPBD) were purified from 293T cell transfectants of their expression vectors with anti-Flag Ab-coated protein A beads. The purity of the Hsp70 and its mutant preparation was analyzed with SDS-PAGE, followed by silver staining. Right panel: effect of Hsp70 mutants (Hsp70ΔABD, Hsp70ΔPBD) on CAD-induced chromatin DNA fragmentation. Assay was performed as described in Figure 4. Percentages of DNA fragmentation calculated as in Figure 2 are shown at bottom. (D) Effect of peptide-binding domain fragment of Hsp70 (Hsp70PBD) on CAD-induced chromatin DNA fragmentation. Assay was performed as described above. Purified Hsp90 was used as a negative control. (E) Cells (5 × 105) of GFP transfectant (GFP-3), Hsp70ΔPBD transfectant, or Hsp70ΔABD transfectant were treated with 1 μM staurosporine (STS) or 10 μg/mL anti-CD3/CD28 Abs, and percentages of apoptotic cell deaths were determined by flow cytometry after propidium iodide staining. The data show the representative results using 2 clones of each transfectant. Each value represents the mean ± SD of 3 independent experiments. **P < .01.

Functional domains of Hsp70 to bind and activate CAD. (A) A scheme of wild-type Hsp70 and its deletion mutants. ATP-binding domain (1-385 amino acids), linker region (385-393 amino acids), and peptide-binding domain (393-640 amino acids) are indicated on the top. ATP-binding domain–deleted Hsp70 mutant (Hsp70ΔABD), peptide-binding domain–deleted Hsp70 mutant (Hsp70ΔPBD), and peptide-binding domain fragment (Hsp70PBD) are shown. (B) Association of CAD with Hsp70 and its mutants. Treated with 1 μM staurosporine were 293T cells that had been transfected with expression vectors for Flag-Hsp70 or its mutants (Hsp70ΔABD, Hsp70ΔPBD, or Hsp70PBD), together with expression vectors for HA-CAD and ICAD, and Hsp70 or its mutants were immunoprecipitated with anti-Flag Ab from cell lysates. Precipitates were immunoblotted with either anti-HA Ab or anti-Flag Ab. To estimate the expression of CAD protein in each cell lysate, an aliquot of each cell lysate was immunoblotted with anti-HA Ab. (C) Left panel: homogeneity of purified Hsp70 mutant proteins. Hsp70 or its mutant proteins (Hsp70ΔABD, Hsp70ΔPBD) were purified from 293T cell transfectants of their expression vectors with anti-Flag Ab-coated protein A beads. The purity of the Hsp70 and its mutant preparation was analyzed with SDS-PAGE, followed by silver staining. Right panel: effect of Hsp70 mutants (Hsp70ΔABD, Hsp70ΔPBD) on CAD-induced chromatin DNA fragmentation. Assay was performed as described in Figure 4. Percentages of DNA fragmentation calculated as in Figure 2 are shown at bottom. (D) Effect of peptide-binding domain fragment of Hsp70 (Hsp70PBD) on CAD-induced chromatin DNA fragmentation. Assay was performed as described above. Purified Hsp90 was used as a negative control. (E) Cells (5 × 105) of GFP transfectant (GFP-3), Hsp70ΔPBD transfectant, or Hsp70ΔABD transfectant were treated with 1 μM staurosporine (STS) or 10 μg/mL anti-CD3/CD28 Abs, and percentages of apoptotic cell deaths were determined by flow cytometry after propidium iodide staining. The data show the representative results using 2 clones of each transfectant. Each value represents the mean ± SD of 3 independent experiments. **P < .01.

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