Journal: Frontiers in Microbiology
Article Title: Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap
Figure Lengend Snippet: Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [ 35 S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C) , separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C) . (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control) . (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.
Article Snippet: Proteins were separated by 10%SDS-PAGE gel, transferred to nitrocellulose and probed in Western blot using anti-DDX3 or anti-eIF specific antibody followed by Alexa Flour 680 goat anti-rabbit IgG (Molecular Probes) or IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) as secondary antibody.
Techniques: Pull Down Assay, Purification, Incubation, In Vitro, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Transfection, Western Blot, Infection, CTL Assay, Confocal Microscopy, Plasmid Preparation, Immunofluorescence, Fluorescence, Staining