Fig. 2.

Induction of fish PKR and PKZ by poly(I · C) and IFN. (A) Schematic illustration of promoter-driven luciferase (Luc) constructs PKRpro-luc (PKRp-luc) and PKZpro-luc (PKZp-luc). (B and C) Activation of the PKR and PKZ promoters by poly(I · C) (polyI:C) or rIFN. CAB cells in each well of 24-well plates were cotransfected with 0.05 μg pRL-TK and 0.5 μg PKRpro-luc or PKZpro-luc in 500 μl medium. At 24 h posttransfection (hpt), the cells were stimulated by transfection of poly(I · C) (0.4 μg/ml) (B) or the addition of rIFN (20 ng/ml) (C). At 48 hpt, the cells were harvested for detection of luciferase activity. Rel. lucif. Act., relative luciferase activity. (D to F) Induction of fish PKR and PKZ proteins by poly(I · C) or rIFN. CAB cells seeded in 6 well-plate were mock transfected with 1.6 μl PBS (D) or transfected with 1.6 μl of a 1-μg/μl poly(I · C) solution (E) or 20 ng rIFN was added to 1 ml medium (F). The cells were harvested at 0, 24, 48, or 72 hpt for detection of PKR and PKZ by Western blot analysis. (G) Requirement of ongoing IFN synthesis for transcriptional induction of fish PKR and PKZ genes. CAB cells seeded in 6-well plates were mock transfected with 1.6 μl PBS or transfected with 1.6 μl of a 1-μg/μl poly(I · C) solution in 1 ml medium or treated with rIFN at 20 ng/ml in the absence or presence of 8 μg/ml cycloheximide (CHX) for 24 h, and total RNA was extracted. Reverse transcription-PCR (RT-PCR) was employed to detect PKR and PKZ expression. (H) Involvement of the Stat1 pathway in PKR and PKZ induction. CAB cells stably transfected with pcDNA3.1 or Stat1-ΔC were treated with 5 ng/ml of rIFN for 10 h and then harvested for detection of PKR and PKZ protein by Western blot analysis.