Figure 6
From: Structure-based drug discovery for combating influenza virus by targeting the PA–PB1 interaction
PA-49 suppresses the expression of viral proteins. MDCK cells (2 × 105 cells/well) were seeded in 24-well plates and infected with A/WSN/33 at an MOI of 1 in the absence or presence of PA-49 (0.5–20 μM). (a) At 9 hpi, the cells were observed by phase contrast microscopy. Scale bar, 150 μm. (b) At 3 or 9 hpi, the cells were then lysed and subjected to 10% SDS-PAGE, and then transferred onto a polyvinylidene fluoride membrane. The membrane was incubated with a 1:30,000 dilution of anti-NP or anti-M1, 1:5,000 dilution of anti-HA or anti-PA, 1:3,000 dilution of anti-PB1, or 1:150 dilution of anti-actin for 4 h, followed by treatment with the biotinylated secondary antibody and streptavidin alkaline phosphatase, and visualisation using BCIP and NBP. (c) The band intensity was quantified by Image J software. The expression level of actin was used for normalisation. To calculate the relative intensity (%) for 3- and 9-h PA-49-treated samples, the band intensity of 9-h- infected cells without PA-49 treatment was shown as 100%. Data represent the means and standard deviation from two independent experiments.
