Figure 3
From: Structure-based drug discovery for combating influenza virus by targeting the PA–PB1 interaction
Effect of PA-49 on subcellular localisation of PA protein. In 24-well plates, HeLa cells were seeded on coverslips and grown to 70–80% confluence. Cells were then transfected with 0.5 μg of plasmid as indicated then 3.5 h after transfection, PA-49 was added then incubated further 25.5 h. Cells were fixed with 4% paraformaldehyde for 10 min, and were permeabilised by 0.1% NP40 in PBS for 20 min. The cells were incubated with 1% skim milk in PBS for 40 min. The cells were then reacted with anti-PA or -PB1 antibody. After washing, the cells were reacted with Alexa Flour 488-conjugated anti-rabbit IgG (Thermo Fisher Scientific K.K., Kanagawa Japan; A11008). (a) Subcellular localisation of PA and PB1 in the absence or the presence of 40 μM PA-49 were shown. Scale bar; 25 μm. (b) The percentage of cells showing greater nuclear than cytoplasmic localisation (N>C), nuclear equal to cytoplasmic localisation (N=C) or cytoplasmic localisation (N<C) was determined by direct counting. The mean and standard deviation obtained from three different areas were shown.
