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. 2012 Nov 29;8(11):e1003059. doi: 10.1371/journal.ppat.1003059

Figure 2. Influenza A Virus NS1 does not interact with mouse TRIM25.

Figure 2

(A) WCLs of human (HEK293T) or mouse (Hepa1.6) cells, that had been mock-treated or infected with PR8 virus at MOI 2 for 18 h, were subjected to IP with or without anti-NS1 antibody, followed by IB with anti-TRIM25. Expression of NS1 and endogenous TRIM25 was determined by IB with anti-NS1 or anti-TRIM25 antibody. (B) Mouse Hepa1.6 or human HEK293T cells were transfected with empty vector, V5-tagged mouse TRIM25 (V5-mTRIM25), or V5-tagged human TRIM25 (V5-hTRIM25), together with NS1-PR8. At 30 h posttransfection, WCLs were prepared and subjected to IP with anti-V5 antibody, followed by immunoblotting with anti-NS1 or anti-V5. (C) In vitro binding of NS1 with TRIM25. Maltose-binding protein (MBP), GST, and the recombinant fusion proteins MBP-human TRIM25-Flag (MBP-hTRIM25-Flag), MBP-mouse TRIM25-Flag (MBP-mTRIM25-Flag), and GST-NS1 (PR8) were purified from bacteria. Purified MBP-hTRIM25-Flag or MBP-mTRIM25-Flag was incubated with GST-NS1. As controls for binding, purified MBP was incubated with GST-NS1, and MBP-hTRIM25-Flag was incubated with GST. Protein complexes were then precipitated with Amylose agarose gel, and precipitates resolved by SDS-PAGE, followed by Coomassie staining. Asterisk indicates the main degradation product of MBP-hTRIM25-Flag.