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. 2009 Jun 12;5(6):e1000473. doi: 10.1371/journal.ppat.1000473

Figure 3. NS1 WT but not dsRNA-binding deficient NS1 protein co-sediments with PKR and vRNP upon density gradient centrifugation.

Figure 3

(A) A549 cells were infected with WT virus or mutant virus #4 at an MOI of 1. Cells were lyzed 12 hours p.i. and subjected to centrifugation through a continuous 5 to 50% sucrose gradient. 16 fractions were taken from top to bottom. Fractions 1 to 9 were analyzed by immunoblotting with antibodies specific for PKR and the viral NP and NS1 proteins. Also, RNA was extracted from gradient fractions 1 to 9 and was subjected to dot blot hybridization with probes specific for HA vRNA and NS vRNA, respectively (panels “HA and NS vRNA”). Whole cell lysates were analyzed by immunoblotting with antibodies specific for phospho-PKR, total PKR, viral NP, viral NS1 and tubulin as indicated (right panel,“lysate”). (B) A549 cells were mock treated or infected with WT virus or virus mutant #4 as described in panel A. Lysates were prepared and subjected to immunoprecipitation with anti-PKR (α) or control antibody (ctrl). The precipitated complexes were analyzed by immunoblotting for PKR and NS1 proteins. RNA was isolated from an identical set of PKR immunoprecipitates of cells infected with the mutant virus and subjected to dot blot analysis with an RNA-probe specific for HA vRNA.