Fig. 5

(A) Co-immunoprecipitation of RC3H1 with NS1 in PR8-infected or mock-infected cells. H1299 cells were either mock or infected with PR8 at MOI of 5 and harvested at 48hpi. Lysates obtained from infected cells were subjected to WB to detect the expression levels of NS1 or RC3H1. Cell lysates were also immunoprecipitated using Protein A/G beads with NS1 monoclonal antibody and the bound proteins were eluted for WB analysis. To resolve the RC3H1 (MW ~ 126 kDa) and NS1 (MW ~ 26 kDa) proteins on SDS-PAGE, different percentages of acrylamide were used and the eluate from the Protein A and G beads was divided based into 2 portions (1:4 ratio) for detecting of NS1 and RC3H1 respectively. (B) and (C) H1299 cells were transfected with 40nM of siRNA for ~ 24 h followed by reseeding into 12-well plates. After another ~ 24 h, the cells were then infected with PR8 virus at MOI of 1. At 15 hpi, the cell culture supernatant was then collected for plaque assay on MDCK cells. A separate set of reseeded cells were harvested for WB analysis to verify the knockdown efficiency. (B) WB analysis showing the expression of RC3H1 in RC3H1 siRNA-treated and negative control siRNA-treated H1299 cells. (C) The amounts of virus produced by RC3H1 siRNA-treated and negative control siRNA-treated H1299 were compared. The average viral titer from 3 independent experiments was plotted and p value was calculated using Student’s t test (***p ≤ 0.001)