Effect of depsipeptide on c-FLIP in CLL cells. Following in vitro exposure to depsipeptide, c-FLIP decreases in CLL cells. CLL patient cells were incubated with or without depsipeptide (DDP) for 4, 24, and 48 hours. (A) Protein lysates were prepared at each time point, separated on a 14% SDS-PAGE gel, and immunoblotted with polyclonal anti–c-FLIP. Gel loading equivalence was confirmed by blotting with an antibody for constitutively expressed protein beta-actin. FLIP expression in these samples was measured by laser densitometry, and is shown relative to the 4-hour untreated sample after equalizing to actin. The positive control (+) is lysate from CLL cells stimulated with CD40L for 12 hours. (B) Prior to lysing, an aliquot of cells from each condition was assessed for early apoptosis by flow cytometry with annexin-V FITC and propidium iodide.