FIGURE 3.

Caspase-8 activity is essential for the cleavage of IRF-3. A, P2.1 cells expressing IRF-3, were pretreated with the inhibitors of multiple caspases (Z, z-VAD, 10 μm), caspase-1 (z-WEHD, 10 μm), or caspase-8 (z-IETD, 10 μm) for 1 h, when the cells were either treated (T) or transfected (R) with poly(I:C) for 8 h, and cell lysates were analyzed by Western blot for IRF-3; B, P2.1 cells expressing Wt IRF-3 were treated with Staurosporine (Stau, 0.5 μm) or Doxorubicin (Doxo, 1 μm), agents that are known to activate caspase-8, or MG132 (10 μm) in the absence or the presence of transfected poly(I:C), and cell lysates were analyzed for IRF-3 by Western blot; C, IRF-3 was isolated from P2.1 cells expressing Flag-tagged Wt human IRF-3 and subjected to in vitro cleavage assay in the presence of varying amount of active caspase-8 (Cas-8) for the indicated times; the reaction mixtures were analyzed by Western blot for IRF-3, using anti-Flag.