Figure 3. HCMV-induced alterations in polysome profile of select mRNA targets results in altered steady-state protein levels in infected cells.
(A) NHDFs were mock-infected or infected with HCMV (MOI=3). At 48 hpi, total protein was collected, fractionated by SDS-PAGE and analyzed by immuno-blotting with the indicated antisera. Akt served as a loading control (LC). (B) Triplicate samples as in A were quantified by immunoblotting using the indicated primary antibodies and a secondary antibody covalently linked to an infrared fluorophore. The membrane was scanned and fold-change upon HCMV infection quantified using an Odyssey infrared imager. Each band was measured for raw intensity value and normalized to the loading control. (C) As in A except total protein was collected from NHDFs that express UL38 from a dox-inducible promoter 72 h post-treatment +/− dox. The LC for uninfected cells was actin. (D) Triplicate samples from C were quantified as described in B to determine the fold change upon UL38 induction in uninfected cells. (E) Average fold-induction of protein accumulation at 72 hpi of select upregulated cellular factors revealed by Power Blot analysis (Stanton et al., 2007) plotted alongside the average fold-recruitment of those representative mRNAs enriched onto polysomes at 48 hpi.