Figure 2
From: Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export
NP mRNA analysis reveals a cell type difference in dependence of Nxf1-mediated nuclear export. A549 and 293 T cells were transfected with plasmid to express dominant negative Nxf1 (DN) or vector control (vec), infected with influenza A Udorn at 2.5 MOI 48 hours post transfection, and fractionated 3.5 hours post infection. A. Cytoplasm and nuclear protein fractions were separated by SDS-PAGE and subject to Western blot to detect SP1, TAT-SF1, or tubulin. Shown is a representative blot from one biological trial. B. RT-qPCR of RNA isolated from the cytoplasm fraction. RNA was quantified and equal concentrations subject to RT with oligo dT. Gene specific PCR was performed using primers to amplify NP, PA, PB1 and PB2 as indicated. Data shown is from two biological independent trials of more than 5 repeats, each trial performed in triplicate PCR. Delta Ct was calculated to determine relative RNA expression. Raw CT values were analyzed in Microsoft Excel using 2ΔCt(average control- average treated). Standard error was obtained by calculating the standard deviation of the sample set divided by the square root of the sample set size, and indicated using error bars. Significance was determined using a two-tailed T-Test conducted in Microsoft Excel, and judging any p value less than .05 as significant, indicated by an asterisk.