Attenuation of B-CLL apoptosis by BLyS.

B-CLL cells from 3 patients (nos. 1-3) were cultured in phenol red-free RPMI supplemented with 0.5% BSA and 0.1 μg/mL Flag-BLyS or 10 μM chlorambucil at 37°C for 18 hours. (A) Caspase-3 activity was determined using the fluorescent caspase-3–specific substrate Ac-DEVD-AMC as described in “Materials and methods.” Apoptosis, as measured by caspase-3 activity, is detected by cleavage of Ac-DEVD-AMC and subsequent fluorescence. The caspase-3–specific inhibitor was included in all conditions to demonstrate specificity. (B) PARP degradation was assessed by Western blot B-CLL cells, stimulated as described above, were directly lysed, separated by SDS-PAGE, and proteins were transferred to an Immobilon P membrane. Membranes were incubated with 1 μg/mL anti-PARP followed by HRP-linked goat antimouse secondary antibody.