Figure 6.

p22-FLIP is a key mediator of NF-κB induction. (A) Primary human T and B cells were stimulated with 1 μg/ml PHA. Primary immature iDCs were stimulated with 500 ng/ml LPS. Western blot analysis was performed using the anti-FLIP mAb NF6, the anti–caspase-8 mAb C15, and the anti-ERK1 mAb. (B) 10⁷ primary human T cells were stimulated with 1 μg/ml PHA for 12 h and lysed, and anti-IKKγ immunoprecipitation was performed. Western blot analysis was performed using anti-IKKα/β antibody and anti-FLIP mAb NF6. (C) Primary human T cells were transiently transfected with double-stranded siRNA oligonucleotides comprising a FLIP-specific sequence (FLIP-1) or a nonspecific sequence (Ctrl.). 48 h after transfection, cells were stimulated with 1 μg/ml PHA for 3 d (right) or 0.5 μg/ml anti-CD3/28 for 24 h (left) or 3 d (right). After incubation, cells were lysed and Western blot analysis was performed using the anti-FLIP mAb NF6 (left), or the proliferation was measured after incorporation of tritiated thymidine ([³H]TdR) during the last 18 h.