10.1371/journal.ppat.1003059.g006 Ricardo Rajsbaum Ricardo Rajsbaum Randy A. Albrecht Randy A. Albrecht May K. Wang May K. Wang Natalya P. Maharaj Natalya P. Maharaj Gijs A. Versteeg Gijs A. Versteeg Estanislao Nistal-Villán Estanislao Nistal-Villán Adolfo García-Sastre Adolfo García-Sastre Michaela U. Gack Michaela U. Gack Influenza A Virus NS1 interacts with mouse Riplet. Public Library of Science 2012 ns1 interacts 2012-11-29 01:44:13 Figure https://plos.figshare.com/articles/figure/_Influenza_A_Virus_NS1_interacts_with_mouse_Riplet_/206253 <p>(<b>A</b>) At 30 h posttransfection with Myc-tagged mouse Riplet (Myc-mRiplet) together with NS1-PR8, HEK293T cells were mock-treated or infected with SeV (10 HA units/ml) for 10 h. V5-tagged hTRIM25 was co-transfected as positive control. WCLs were subjected to IP with anti-Myc (Riplet) or anti-V5 (TRIM25), followed by IB with anti-NS1 antibody. (<b>B</b>) Hepa1.6 cells were transfected with Flag-tagged mouse Riplet. At 30 h posttransfection, cells were either mock-treated or infected with recombinant A/PR/8/34 virus at an MOI of 2. 18 h later, WCLs were subjected to IP with anti-NS1 antibody, followed by immunoblotting using the indicated antibodies. (<b>C</b>) Localization of NS1-PR8 and mouse Riplet in HeLa cells determined by confocal microscopy. At 30 h posttransfection with Myc-mRiplet alone, NS1-PR8 alone, or NS1 together with Myc-mRiplet, HeLa cells were stained with anti-Myc (green), anti-NS1 (red), and DAPI (nucleus, blue). Fifty cells from three independent experiments were counted and the percentage of cells with cytoplasmic NS1 is shown, ***p<0.001 by Fisher's exact test. (<b>D and E</b>) Hepa1.6 cells were transfected with Flag-tagged mouse Riplet. At 30 h posttransfection, cells were either mock-treated or infected with recombinant A/PR/8/34 virus expressing NS1 PR8, Cal04, HK156, Tx91, SwTx98, or Pan99 at an MOI of 2 (<b>D</b>), or R38A/K41A or E96A/E97A NS1 mutant at an MOI of 4 (<b>E</b>). 18 h later, WCLs were subjected to IP with anti-NS1 antibody, followed by immunoblotting using the indicated antibodies.</p>