Figure 3

Nxf1 is associated with NP, HA, and NA influenza mRNAs in A549 cells. A549 cells were transfected with plasmid to express FLAG-Nxf1 or FLAG-Vector control and infected with influenza A Udorn at 2.5 MOI 48 hours post transfection. Cells were collected 7 hours post infection and total extract prepared and subject to immuno purification using anti-FLAG antibody coupled to beads. Protein and RNA were isolated from total extract, immuno purified beads, and supernatant. A. Protein from the immuno purified beads (IP) and supernatant (sup) were separated by SDS-PAGE and subject to Western blot with anti-FLAG antibody to detect FLAG-Nxf1. Shown is a representative blot from one biological trial. B-C. RNA isolated from IP and total samples was quantified and equal concentrations were subject to RT with oligo dT. Gene specific PCR was performed using primers to amplify NP, PA, PB1, PB2, HA, and NA for 25 cycles. Data shown is from two representative trials of more than 5 repeats. In all cases the volume of cDNA used in the PCR step was 1/10^th that used in the IP samples, with the exception of NP amplification in B, where equal cDNA was used for total and IP, and total samples are likely at saturation limit in end point PCR.