The 103L and 106I residues in the H5N1- NS gene are associated with increased cytoplasmic localization of NS1 protein in mouse epithelial cells. Monolayers of mouse epithelial cells were infected with rPR8-H5N1-NS-103L+106I (n=3) and the three mutants rPR8-NS-L103F+106I, rPR8-NS-103L+I106M and rPR8-NS-L103F+I106M (n=2) at MOI=2 and compared to mock uninfected cells. NS1, NP and M1 proteins localization was detected by western blots using rabbit protein specific antibodies. Rabbit anti-tubulin and mouse anti-histone antibodies were used as controls for cytoplasmic and nuclear fractions respectively. a. The nuclear and cytoplasmic accumulation of NS1 protein is shown for one analysis. The whole cell lysate is designated as “W”, nuclear fraction as “N” and the cytoplsmic fraction as “C”. b. Western blots showing the H5N1-NS1 protein nuclear and cytoplasmic fractions in parallel for 2–3 replicates of infected M1 cells.