Fig. 5.

Titration of NP and reconstituted vRNP activity. 293 T cells were transfected with plasmids to express reconstituted vRNPs with GFP-M vRNA and either WT-NP, no NP, or NPbd3 at concentrations indicated. a Total protein from titration samples was isolated at 48 h post transfection and run on a 10% SDS PAGE and transferred to nitrocellulose. The membrane was probed with FLAG to identify NP and Tubulin as the loading control. Expected size was confirmed by comparison with Fisher BioReagents EZ-Run protein standards. b Cells were observed for GFP-M expression 48 h post transfection. WT-NP expressed by transfection of 400 ng plasmid is the standard concentration used in our reconstituted vRNP experiments and serves as positive control, while no NP is the negative control. GFP was visualized with a Nikon Eclipse TS100 (Nikon Intensilight C-HGFI for fluorescence) inverted microscope and images captured with the Nikon DS-Qi1Mc camera with NS Elements software