FIG. 4.
PKRwt and mutant PKR/KR296 can activate NF-κB in EMSAs. (A) PKR0/0 cells were transfected with 250 ng or 1 μg of plasmid pcDNA1/Amp expressing PKRwt or PKR/KR296 as described in Materials and Methods. The total amounts of plasmids were adjusted to 10 μg with the vector pcDNA1/Amp. All plasmids were prepared with an Endo-free Plasmid kit (Qiagen). Positive controls for NF-κB activation were obtained by transfecting 10 μg of pCMV-Tax (TAX). Background NF-κB activity is shown for cells transfected with pcDNA1/Amp alone (pc/Amp). Five micrograms of nuclear extracts was incubated with 0.2 pmol of 32P-labeled NF-κB probe for 20 min in the presence of 1 μg of poly(dI-dC) · poly(dI-dC) at 20°C and analyzed by an EMSA on 5% native acrylamide gels. Inducible NF-κB complex is indicated by the arrow. (B) Five micrograms of nuclear extracts from PKR/KR296-transfected cells was incubated for 10 min either as such (−) or in the presence of anti-p50 antibody (Ab) (shift indicated by upper arrow) or anti-p65 antibody (disappearance of the complex) prior to the addition of the probe and processed as described above.