FIG. 3.
PKR/wt and mutants PKR/KR296 and PKR/Del42 stimulate NF-κB-dependent reporter gene expression in PKR0/0 MEFs. (A) The assay was performed as described in the legend to Fig. 1, except that PKR0/0 MEFs were used instead of PKR+/+ MEFs. The reporter plasmids pHIV-1 LTR-luc (dotted bars) and pHIV-1 LTRΔNF-κB-luc (grey bars) (100 ng each) were transfected either as such (0) or in the presence of 1 or 200 ng of PKRwt, PKR/KR296, or PKR/Del42. Data (luciferase [Luc] activity) are expressed as the mean of four independent transfections ± the standard error. (B) RT-PCR analysis of total RNA from PKR0/0 MEFs (in 10-cm petri dishes) transfected with pGL2 HIV-1 LTR-luc (LTR-Luc) alone or in the presence of increasing concentrations of PKRwt (0.1, 1, and 10 μg of plasmid). The sizes of the PCR products were estimated with a φX174 DNA HaeIII digest as a marker. The upper band (561 bp) corresponds to amplification from the transfected DNA as compared to the size of the PCR product from the pHIV-1 LTR-luc plasmid (P). The lower band (497 bp) is specific for DNA amplified from the reverse-transcribed luciferase mRNA. Control GAPDH RT-PCR is shown below. (C) RT-PCR analysis of total RNA from PKR0/0 MEFs transfected as described for panel B with the vector (LTR-Luc) alone or in the presence of PKR/KR296 or PKR/Del42 (0.1 and 10 μg of plasmid).