Confirmation of the viral infection and cellular fractionation procedures prior to LC-MS/MS analysis.

<p>(A) A549 cells were infected with DENV-2 at a m.o.i. of 5. Samples of the culture supernatant were taken over the period 24–96 hours post-infection and assayed for the presence of infectious virus by immunofocus assay. The titers are shown as focus forming units (FFU) per ml and are an average of two independent experiments with error bars shown. (B) SILAC labeled A549 cells that had been either DENV-2 (DV2) or mock (Mo) infected at a m.o.i. of 5 were detached with trypsin at 28 hours p.i. and harvested prior to fractionation. Samples of the detached cells were applied to glass coverslips, fixed with methanol and examined for the presence of the DENV-2 E protein by immunostaining using a specific antibody (anti-E) (right-hand panels) or observed under the light microscope (left-hand panels). (B) Western blot analysis of cytoplasmic (Cyt) and nuclear (Nuc) fractions prepared from the SILAC labeled A549 cells infected with DENV-2 or mock infected. Ten μg of protein from each cellular fraction was analyzed for the presence of the DENV-2 E and NS5 proteins and the cellular proteins lamin A/C, β-tubulin and GAPDH using specific anti-sera. The results shown are typical of two independent Western blotting experiments. The positions of relevant molecular mass markers are shown in kDa.