Absence of PKR-dependent NF-κB activation in 3T3 cells deficient in TRAF2 and TRAF5. (A) 3T3 cells grown in 10-cm-diameter plates, obtained from WT mice (WT 3T3) or mice deficient in both TRAF2 and TRAF5 (DKO 3T3), were mock infected (M) or infected with 5 PFU of VV per cell (VV) or 5 PFU of VV PKR per cell (PKR). Nuclear extracts were prepared at 20 hpi and analyzed by gel shift assay with a probe specific for NF-κB. (B) Extracts (50 μg) from the same cells were analyzed for PKR expression by Western blotting (WB). (C) Cells infected as described for panel A were analyzed for induction of apoptosis as indicated in Materials and Methods. The lane numbers in panels B and C are the same as those in panel A. O.D., optical density. DKO (D) or WT (E) 3T3 cells were treated with 10 μg of pIC per ml for the times indicated, and levels of IκBα and β-actin were analyzed by Western blotting. The arrows indicate specific proteins.