Figure 2

Expression from capped S10-GFP RNA is upregulated by NS1. (A) BSR 96 well monolayers transfected with 100 ng BTV pCAG clones or empty pCAGGS vector together with 100 ng capped S10-GFP reporter RNA. Fluorescence was recorded at 20 h after transfection with capped S10-GFP reporter RNA. (B) Dose–response relationship of expression from capped S10-GFP RNA across a two-fold dilution series of NS1 expression. BSR 96 well monolayers transfected with 0 ng - 200 ng pCAG NS1 and the capped S10-GFP reporter RNA. Fluorescence was recorded at 20 h after transfection with S10-GFP reporter RNA at low magnification (10x objective lens) to maximise the number of cells within the field of view. Values indicate the number of fluorescing cells within the field. (C) Expression of NS1 in BSR 96 well monolayers transfected with the same quantities of pCAG NS1 used in panel B. Transfected monolayers were harvested at 18 h after transfection and analysed by SDS-PAGE, followed by immunoblotting using anti-NS1 rabbit polyclonal antibody R633. The quantities above the lanes indicate the amount of plasmid transfected. NS1 positive control -2 μg BTV-10 NS1 purified from Sf9 cells infected with recombinant baculovirus Ac BTV NS1. The numbering on the left indicates the molecular weight of the size markers.