Fig. 3.

Concomitant induction of eIF2α phosphorylation by poly(I · C) and IFN. (A to C) Concomitant induction of eIF2α phosphorylation by poly(I · C) and rIFN. Samples loaded here are the same as those in Fig. 2D to F, respectively, but phosphorylated eIF2α (p-eIF2α) and total eIF2α were detected by Western blot analysis. (D) Involvement of the Stat1 pathway in eIF2α phosphorylation by poly(I · C) and rIFN. The same samples shown in Fig. 2H were loaded here for Western blot analysis of total eIF2α or phosphorylated eIF2α. (E) Direct interaction of eIF2α with PKR or PKZ. CAB cells seeded in 10-cm dishes were mock induced by transfection of 8 μl PBS [poly(I · C) absent] or induced by transfection of 8 μl × 1 μg/μl poly(I · C) [poly(I · C) present] in 5 ml medium for 48 h and then harvested for coimmunoprecipitation assay by anti-eIF2α antibody. PKR, PKZ, p-eIF2α, and eIF2α were analyzed by Western blotting in both immunoprecipitation (IP) samples (left blots) and input samples (right blots). (F to H) CAB cells were transfected with poly(dG · dC) which mimics Z-DNA (F), poly(dA · dT) which mimics B-DNA (G), or unsheared calf genomic DNA (g-DNA) (H), harvested at 0, 24, 48, and 72 h posttransfection (hpt), and subjected to immunoblotting with antibodies specific to PKR, PKZ, actin, p-eIF2α, and eIF2α. The relative levels of eIF2α phosphorylation as determined by quantitative densitometry are shown below the blots. The value for the sample at 0 hpt is set at 1 as a reference with which other samples are compared.