ApoL9 proteins are not-secreted, cytoplasmic proteins.

<p>A. Antiviral activity of N-terminally (FLAG-ApoL9b) and C-terminally (ApoL9b-FLAG) FLAG-tagged ApoL9b against TMEV. Histograms show the relative infection efficiency, of L929 cells that were transduced with theTM945 lentiviral vector expressing mCherry alone (vector), or with the MK82 or MK83 derivatives co-expressing mCherry and FLAG-ApoL9b or ApoL9b-FLAG, respectively. Infection efficiency was measured, by flow cytometry, after infection of cells for 10 hours with 0.5 PFU per cell of the GFP-expressing KJ26 virus. B. FLAG detection (green) and nuclear staining (blue) in HeLa-M cells transduced with the MK65 construct expressing FLAG-ApoL9b (scale bar: 10 um). Typical immunolabeling patterns are shown: left and central panels: one non-transduced cell (arrow) and transduced cells showing a diffuse cytoplasmic FLAG-ApoL9b localization; right panel: cells showing typical small and medium-sized FLAG-ApoL9b granules (arrows). C. Confocal images showing FLAG (green—left panel) and mitochondria (red—central panel) labelings and merged image (right panel). Nuclei were stained with Hoechst (Blue). D. Western blot detection of FLAG-tagged ApoL9b, ApoL9a and IFN-αA in 293T cells. Cells were mock-transfected, transfected with the empty pTM943 plasmid (vector), or with pTM943 derivatives coding for the indicated FLAG-tagged ApoL9 proteins. As a control cells were also transfected with plasmid pcDNA3-IFNαA-FLAG, which expresses the secreted FLAG-tagged mouse IFN-αA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133190#pone.0133190.ref012" target="_blank">12</a>]. 20 hours post-transfection, cells were mock-treated (-) or treated for 4 hours with 10ug/ml of Brefeldin A (+) prior to total protein extraction. FLAG and β-actin were detected by Western blot in cell lysates.