Pronounced up-regulation of the short isoforms of c-FLIP in CD40-activated ALL blasts mediates resistance to death receptor–dependent apoptosis. (A) For assessment of expression levels of molecules that form the death-inducing signaling complex (DISC), cell lysates of resting and CD40-activated normal B cells and ALL blasts were analyzed by Western blotting. (B) The relative expression of c-FLIP_S+R and c-FLIP_L proteins in CD40-activated normal B cells and ALL blasts was measured by densitometry using Image Java software program (NIH, Bethesda, MD). Results of samples from 5 individual patients and 4 healthy B-cell controls are represented as the mean ± SEM of FLIP_S+R/FLIP_L peak area ratio. (C-D) CD40-activated ALL cells were treated with anti-CD95 mAb in the absence or presence of 0.1 μg/mL cycloheximide (CHX). After 48 hours, expression of the long and short isoforms of c-FLIP was examined by Western blot analysis (C), and CD95-dependent apoptosis was measured by flow cytometry (D). Values represent mean percentage of specific apoptosis (± SEM; n = 7 patients) (t test ± CD40L, P = .004; t test CD40L ± CHX, P = .01).