Activation of caspases-3 and -8 and cleavage of PARP in CD40-activated CLL cells. CLL cells were activated via CD40 ligation. Twenty-four hours later, these cells were treated without or with 1540-14 at 2 different concentrations and/or CH11, either alone or in combination, as indicated at the bottom of the figure. Incubation with the combination was done with or without the nonspecific caspase antagonist zVAD-fmk. After either 6 or 12 hours, the CLL cells were harvested and used to prepare lysates for immunoblot analysis. (Upper panel) Caspase-3 activity was detected by the addition of the DEVD substrate to 10 μg total protein lysates. The assay was performed measuring the release of fluorescent dye using a fMax spectrofluorometer plate reader (excitation at 485 nm and emission at 538 nm). Mean data are presented of 3 independent experiments ± SE. Y-axis represents the V_max. (Lower panel) Protein lysates were prepared and normalized for total protein content. Cell lysate (15 μg) was applied to each lane of a polyacrylamide gel for immunoblot analyses with anti-caspase-8 (first row), anti-caspase-3 (second row), anti-PARP (third row), anti-XIAP (fourth row), or anti-β-actin (fifth row), as indicated on the left margin of the figure.