<p>(A) The entire M1 RNA was radiolabeled uniformly at either C (left) or U (right) residues, incubated with nuclear extract under splicing conditions, crosslinked with 254 nm light and digested with RNase. The reaction was then either resolved by SDS-PAGE (10% Total) or incubated in separate reactions with the antibodies indicated (IP: anti-). Immunoprecipitated proteins were resolved by SDS-PAGE. Position of molecular weight markers are also shown. Vertical dotted line shows that the samples were loaded in the same gel but not next to each other. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003460#ppat.1003460.s004" target="_blank">Figure S4</a> shows immunoblot analysis of nuclear extract with antibodies against NS1-BP and hnRNP U, which did not directly bind M1 mRNA but are present in the extract. (B) A549 cells were infected with A/WSN/33 at MOI 5 for 5 h and were then cross-linked with 0.3% formaldehyde. Cells were lysed with RIPA buffer, and the lysates were immunoprecipitated with control IgG or NS1-BP antibodies. After a series of washes, the cross-link was reversed, and associated RNAs were isolated. Real-time quantitative RT-PCR was used to measure M1 mRNA. Housekeeping genes (GAPDH, β-actin, and α-tubulin) were included to test the specificity of the assay. Data was normalized to the viral M segment. The graph displays the mean ± SEM (n = 3). Comparison between cross-linked samples shows significant (p<0.05) differences between association of NS1-BP with M1 mRNA and the lack of association of NS1-BP with the 3 host mRNAs tested.</p
