Figure 6.

Caspase activation results in selective substrate cleavage. Total proteins from 10⁶ unstimulated (−) or anti-CD3–activated (+) PBMCs were obtained at different time points (day 1 to day 4), separated by SDS-PAGE, and analyzed by Western blot using an anti-PARP, DFF45, or caspase-3 antiserum, or an anti-Wee1 mAb. As a control for caspase-mediated cleavage, total proteins from 10⁶ Jurkat cells cultured on uncoated or anti-Fas–coated plates (M3: 20 μg/ml) for 4 h were subjected to the same treatment as PBMCs, and results are shown on the right. The 45-kD strong band observed with the anti-Wee1 antibody throughout the kinetic results from a cross-reactivity observed in some tissues with this antibody, according to the manufacturer. These experiments were performed three times, and gave similar results with different PBMC donors.