Figure 6. The inhibition of luciferase reporter activity is partly dependent on PKR protein level.

(A) Stable cell line with shRNA-mediated PKR knock-down. Western blot analysis of HeLa stable cell lines carrying shRNA vector targeting PKR. Ctrl, parental HeLa cells; A1-B6, stably-transfected independent clones carrying antibiotic resistance. Clone B4 showed the highest PKR knock-down and was used for subsequent experiments. (B) Parental HeLa cells (ctrl) or HeLa cells with stably down-regulated PKR expression (KD) were co-transfected with 100 ng/well of each RL (light colors) and FL (dark colors) reporter plasmids and 0 or 50 ng/well of pCAGEGFP-MosIR. Parental plasmid pCAGEGFP was used to maintain a constant amount of transfected DNA. Data are shown as an average of two independent experiments performed in quadruplicates; Error bars  =  SEM. (C) pCAGEGFP-MosIR transcript associates with PKR. HEK-293 cells were transfected with pCAGEGFP or pCAGEGFP-MosIR. Cell lysates (24 hours post-transfection) were used for immunoprecipitation with anti-PKR antibody or control IgG antibody. Immunoprecipitated RNA was reverse-transcribed and used for real-time PCR analysis. Data are displayed as a percentage of input. Shown is the average of two experiments. Error bars  =  range of values. (D) pCAGEGFP-MosIR expression activates PKR. Western blotting analysis of HEK-293 cells transfected with increasing amount of pCAGEGFP or pCAGEGFP-MosIR (50–150–250 ng per well). pBluescript was added to maintain the amount of transfected DNA constant. pBS, pBluescript only. UN, untransfected cells; MosIR, pCAGEGFP-MosIR.