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. 2017 Mar 21;8:14751. doi: 10.1038/ncomms14751

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Copyright © 2017, The Author(s)

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(a) HEK293T cells were co-transfected with FLAG-SF2 and V5-NS1 or V5-vector, and cell lysates used for IP with α-V5 or control IgG, followed by immunoblotting (IB) with α-FLAG and α-V5. (b) A549 cells were infected with rH9N2-WT viruses at an multiplicity of infection (MOI) of 5 for 16 h and cell lysates used for IP with α-SF2/ASF (endogenous) or control IgG, followed by IB with α-SF2/ASF and α-NS1. (c) HEK293T cells were co-transfected with V5-NS1 or V5-vector, along with FLAG-SF2ΔRS plasmids and cell lysates were then used for IP with α-V5 or control IgG, followed by IB with α-FLAG and α-V5. (d) HEK293T cells were transfected with FLAG-SF2, together with V5-NS1, V5-RBD of NS1, V5-ED of NS1, V5-tagged mutant NS1-R38A/K41A (38A/41A) or V5-vector, and cell lysates were used for IP with α-V5 or control IgG (control IgG blots not shown), followed by IB with α-FLAG and α-V5. (e) HEK293T cells were co-transfected with FLAG-SF2 and V5-NS1, with cell lysates being immunoprecipitated with α-V5 or control IgG and washed, then treated or mock-treated with RNase A at 37 °C for 45 min and further washed, followed by immunoblotting with antibodies against FLAG or V5. Co-transfection of HA-RHA (RNA Helicase A) and V5-NS1 is used as a control to demonstrate an RNA-dependent interaction. (f) Bacterially expressed GST-SF2, GST (control) and dual His-tagged NS1 proteins were purified and used in a GST pull-down assay, followed by IB with α-His and α-GST. A Coomassie blue-stained SDS–PAGE gel shows the purity of recombinant proteins. Full-size western blottings are provided in Supplementary Fig. 8.