In vitro kinase assay

The tested proteins TYK2, HA-STAT1, HA-STAT2 or Myc-NS4A in Figure 6(A) were in vitro translated with a TNT Quick-coupled Transcription/Translation Systems kit (Promega) following instructions of the manufacturer. Kinase reactions were performed by incubation of 1.0 μg purified HA-STAT1 or HA-STAT2 as the substrate with 1× kinase buffer, 1 mM ATP, immunoprecipitated Myc-NS4A and JAK1 or TYK2 at 30°C for 60 min in 50 μl reaction mixture. Reaction was stopped by addition of 2×SDS loading buffer and samples were separated by SDS-PAGE, and analysed by immunoblotting with anti-phospho-STAT1 or anti-phospho-STAT2.

NS4A interacts with the SH2 and TAD domain of STAT1 or STAT2 to impair their phosphorylation. (A) In vitro kinase assay of STAT1 phosphorylated at Tyr701 (Y701) and STAT2 phosphorylated at Tyr690 (Y690). The peptide of STAT1 or STAT2 and their kinase JAK1 or TYK2 were introduced into a mixture containing Flag-NS4A. (B) Schematic representation of STAT1 or STAT2 (top). IB analysis of HEK293 T cells co-transfected for 48 h with Flag-NS4A and HA-wild-type STAT1 or STAT2, or their mutants, assessed with whole-cell lysates (10% input) or IP with anti-HA antibody (bottom). The numbers in A means the relative intensity of the p-STAT1/ STAT1 or p-STAT2/STAT2 detected by Western blotting. Data are representative of three independent experiments.