Figure 3

Impact of RNA binding domain and effector domain of NS1 protein on dsRNA induced NF-κB promoter activity in A549 and MiLu cells. (A) A schematic presentation for the construction of the RNA binding domain, the effector domain from both alleles of H6N8 and chimeric NS1s. (B) A549 cells were transiently co-transfected with NF-κB luciferase reporter plasmid, and vector encoding NS1 from H6N8-A, H6N8-B, H6N8-A-RNA, H6N8-A-ED, H6N8-B-RNA, H6N8-B-ED, H6N8 chiNS1 A/B, H6N8 chiNS1 B/A or empty pcDNA3.1+ vector (pNF-κB cont.) or left un-transfected (cells). 24 hours post transfection, cells were stimulated with dsRNA, cell extracts were prepared and luciferase activities were measured using the ONE-Glo™ Luciferase Assay System (Promega). The values were normalized and pNF-κB cont. was set to 100%. (C) All the conditions in MiLu cells transfection were maintained as practiced in (B). Error bars indicate standard deviations. The data shown are representative for three experiments with transfections performed in duplicate. * indicates a significant difference as determined by the student's t-test, with p-values of < 0.05.