Figure 2.

Activation of purified PKR by in vitro transcribed RNA. Purified PKR was incubated with γ-³²P-ATP and in vitro transcribed mRNA for 10 min. Reaction products were separated by SDS–PAGE and imaged using phosphor storage radiography. Unmodified or Ψ-containing mRNAs encoding firefly luciferase contained triphosphates (ppp) or cap at their 5′-ends. Complete capping of RNA was achieved post-transcriptionally using vaccinia capping enzyme. Concentration of mRNA in reactions was 3.1, 6.2, 12.5 and 25 µg/ml. Quantified phosphorylation is presented as a bar graph below each band. Values were normalized to those obtained with 25 µg/ml uncapped, unmodified RNA. No RNA (−) and 79 bp dsRNA were used as negative and positive controls.