Figure 5.
siRNA silencing of NS3. (A) OK cells were transfected either with a mix of NS3 specific siRNAs or scrambled control siRNA. Seven hours post-transfection, cells were infected with EHDV2-IBAV for an additional 14 h. Cell lysates were separated by 10% SDS–PAGE and immunoblotted with anti-NS3 or anti-NS2 antibodies. The graph depicts average of ± SD of NS3/NS2 immunoblot signal ratio from four independent experiments. *P < 0.0008. (B) OK cells transfected with a mix of NS3 specific siRNAs or scrambled control siRNA were infected with EHDV2-IBAV. 16 h post-infection cell medium was collected, and cells were lysed by sonication. Plaque assay was performed using cell lysates or supernatant. The graph depicts average of plaque forming units (PFU) per well (of 12-well plate) of five independent experiments. *P < 1.2E-11, **P < 1E-10. (C) OK cells transfected and infected as the above, were co-transfected with Cy3- and Rd110-labeled tRNAIleUAU, fixed 6 h post-tRNA transfection and imaged by confocal microscopy. FRET signals were measured and FRETc signals were calculated. Panels show representative cells. Bars are 5 µm. (D) Graph depicts average ± SD of FRETc/Cy3- tRNA fluorescence ratio per cell (n = 30). *P < 1.1E-16, **P < 3E-18.