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. Author manuscript; available in PMC: 2014 Aug 15.
Published in final edited form as: Virology. 2013 May 23;443(1):48–58. doi: 10.1016/j.virol.2013.04.020

FIGURE 3. Effect of PKR on reporter expression mediated by IRES-dependent compared to 5’ cap-dependent translation initiation.

FIGURE 3

FIGURE 3

PKR-deficient U cells (UPKRkd) were co-transfected with monocistronic reporter constructs expressing either firefly (IRES-FF) or Renilla (5’ cap-RL) luciferase, along with either empty vector (Vec) or the indicated DNA amount (µg) of PKR expression plasmid encoding either wildtype (WT), catalytic mutant (K296R), RNA-binding mutant (K64E) or the catalytic and RNA-binding double mutant ( KE-KR) PKR protein. At 24 h after transfection extracts were prepared and analyzed. (A) Relative luciferase activity of PKR-transfected compared to vector-transfected cells; the mean and standard deviation were determined from three independent experiments. The absolute values for Renilla (5’-cap) and firefly (IRES) monocistronic reporter expression were 2.3 × 106 RLU for Renilla and 9.7 × 103 RLU for firefly luciferase in the vector-transfected cells which were normalized to 1.0. (B) Representative western blots showing the levels of PKR, Thr446 phospho-PKR (P-PKR), eIF2α, and Ser51 phospho-eIF2α (P-eIF2α), and β-actin as a loading control.