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. 2014 Aug;88(16):9038–9048. doi: 10.1128/JVI.00830-14

FIG 3.

FIG 3

NS1 interacts with PACT during virus replication and in vitro. (A) HEK293T cells were infected with PR8-wt or PR8 NS-FS for 24 h, followed by immunoprecipitation (IP) and immunoblotting (IB) with antibodies against PACT and NS1 (Ly, total lysate). (B) HEK293T cells were transfected with PACT along with FS-tagged NS1 or NS2 for 48 h, followed by IP/IB analysis using antibodies against FLAG (for IP) and antibodies against FLAG (detecting FS-NS1 and FS-NS2) and PACT (for IB). (C) HEK293T cells were infected with PR8-wt or PR8 NS-FS for 6, 12, or 24 h, followed by IP and IB with antibodies against FLAG (for IP) and PACT and NS1 (for IB). (D) HEK293T cells were transfected with control plasmid or HA-tagged PACT, followed by infection with indicated IAV strains for 24 h and IP/IB analysis using antibodies against HA (for IP) and PACT, NS1, and p38 (for IB). H1N1, PR8; H7N3, A/laughing gull/Delaware bay/42/2006; H4N2, A/quail/California/D113023808/2012; H3N2 (A/Perth/16/2009). (E) Pulldown experiment of in vitro-translated PACT using recombinant, bead-immobilized GFP or NS1. A pull-down sample and 10% of input sample were resolved by SDS-PAGE and analyzed by using a phosphorimager or SYPRO Ruby protein staining, respectively.