Fig. 3.

Interaction of BVDV2a 1373 Npro and S100A9 cellular protein. The interaction of BVDV Npro with S100A9 protein in cells were confirmed by co immunoprecipitation assays. The 293T cells were co-transfected with pEGFP-C1-Npro and p3XFLAG -S100A9 plasmid or pEGFP-C1-Npro and p3XFLAG. At 24 h post transfection, cells were lysed and the proteins were isolated (by co-immunoprecipitation) using the anti-FLAG beads. Isolated proteins were analyzed for presence of GFP (e.g GFP-Npro) as well as FLAG using western blot using anti-GPF or anti-FLAG antibodies. The mock transfected 293T cells were used as negative control while cells transfected with pEGFP-C1-Npro plasmid were used as GFP-Npro positive control. Lane (1) The 293T cells without co-transfection did not show the presence of GFP-Npro or FLAG- S100A9, Lane (2) cells transfected with pEGFP-C1-Npro alone and showed the presence of GFP-Npro, Lane (3) cells transfected with p3XFLAG -S100A9 and showed the presence of S100A9, Lane (4) cells were co transfected with empty p3XFLAG along with pEGFP C1-Npro and did not show the presence of Npro (the FLAG size was 8 aa and was seen at very lower side of the gel). Lane (5) Cell lysates from cells co-transfected with p3XFLAG S100A9 along with pEGFP C1-Npro which showed the presence of S100A9 as well as Npro.