7 research outputs found
Activity of FLAG-tagged and untagged mouse IFNs.
<p>Histograms show the log<sub>2</sub> of antiviral activities detected in the supernatant of Neuro2A cells collected 24h after transfection of the pcDNA3 derivatives expressing the indicated FLAG-tagged (+) or untagged (-) IFNs or after transfection of the corresponding empty vectors (pcDNA3). Antiviral activities are presented relative to those of culture medium. NS: non significant (Mann–Whitney <i>U</i> test).</p
Processing of the IFN-ε precursor in transfected cells.
<div><p>Western blot analysis of IFN-α and IFN-ε processing in total protein extracts of Neuro2A cells transfected for 24h with pcDNA3 derivatives expressing FLAG tagged IFNs.</p>
<p>A. Mouse IFN-αA and IFN-ε detection in the presence or absence of brefeldin A. Arrowheads point to the two bands detected for IFN-ε.β-actin detection was used as loading control. B. Detection of mouse IFN-αA and IFN-ε along with corresponding proteins expressed without signal peptide (Δsp). C. Human IFN-ε and mouse IFN-β detection in 293T cells before and after treatment with N-glycosidase F.</p></div
Plasmid constructs.
<div><p>A. pcDNA3 derivatives expressing FLAG-tagged or untagged IFNs. In these plasmids, IFN coding regions (frames) are cloned downstream of the cytomegalovirus promoter (pCMV). Restriction sites used for cloning IFN reading frames are shown. FLAG tag coding sequences were added downstream of the last IFN codon as indicated in C. IFN-αA(Δsp) and IFN-ε(Δsp) are constructs where the region encoding the signal peptide of the IFN precursor was deleted. lim-ε: chimeric IFN precursor with the signal peptide of limitin and the mature moiety of IFN-ε.ε-lim is the converse chimeric precursor with the signal peptide of IFN-ε and the mature moiety of limitin. Human IFN-ε with or without the signal peptide are indicated hIFN-ε and hIFN-ε(Δsp). Note that the various elements on these graphic representations are not to scale.</p>
<p>B. Lentiviral vectors. In these vectors, transcription of the IFN gene is driven by the cytomegalovirus promoter. The IRES sequence from TMEV allows co-expression of the cloned coding sequence with the red fluorescent protein mCherry.</p>
<p>C. Sequence of the IFN-FLAG junctions. X represents the last amino acid of IFN. The linker sequence between IFN and FLAG (bold letters) is AAA for ε-lim and limitin and TAA for the other constructs.</p></div
IFN-ε signal peptide is not fully functional.
<div><p>A. Signal peptides predicted for IFN-ε and limitin. Predicted signal peptides are indicated in bold letters. Related amino acids around the putative cleavage site are framed.</p>
<p>B. Western blot analysis of cell lysates from Neuro2A cells that were transfected for 24h with pcDNA3 derivatives expressing FLAG-tagged IFN-ε, IFN-ε(Δsp), lim-ε, ε-lim or limitin. Cells were harvested in laemmli buffer twenty-four hours post-transfection.</p>
<p>C. Histograms showing, for the indicated constructs, the proportion of cells where IFN colocalizes mostly with the Golgi or with the endoplasmic reticulum. Means and SD of countings from 4 immunostainings. For each counting, n = ± 200 for IFN-α, lim-ε, limitin and ε-lim; n = 100 for IFN-ε.</p>
<p>D. Immunofluorescent detection of FLAG-tagged IFNs in HeLa cells transfected with plasmids expressing the indicated tagged IFNs. IFNs appear in green. The WGA lectin was used to detect glycosylated proteins and to highlight the Golgi network (red).</p></div
Primers.
a<p>Sequence kindly provided by Professor Stephan Brand, University Hospital Munich-Grosshadern, University of Munich, Germany.</p>b<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087906#pone.0087906-OConnor1" target="_blank">[32]</a>.</p>c<p>(f) forward primer; (r) reverse primer.</p>d<p>For RT-qPCR, annealing reactions were performed at 63°C for influenza A virus and at 60°C for mOASl2, hOAS1, hMxA and β-actin.</p
IFN response in mice infected with the TURH influenza A virus strain.
<p><b>A:</b> Immunofluorescence detection of TURH influenza A virus in liver sections from Mx-IFNAR1<sup>0/0</sup> and Mx-IFNLR1<sup>0/0</sup> mice infected for 48 hours or mock-infected, using anti-influenza A virus antibody (Red) and Hoechst staining of nuclei (blue). Results are representative of two independent experiments. <b>B–C:</b> RT-qPCR analysis of influenza A virus replication in the liver (B) and lungs (C) of Mx-IFNAR1<sup>0/0</sup> and Mx-IFNLR1<sup>0/0</sup> mice. Results are expressed as influenza A virus cDNA copies per 10<sup>5</sup> copies of β-actin cDNA. <b>D–E:</b> RT-qPCR analysis of <i>Oasl2</i> expression in the liver (D) and lungs (E) of Mx-IFNAR1<sup>0/0</sup> and Mx-IFNLR1<sup>0/0</sup> infected mice. Results are expressed as <i>mOasl2</i> cDNA copies per 10<sup>3</sup> copies of β-actin cDNA. <b>F–G:</b> IFN-β (F) and IFN-λ (G) production in the liver of infected mice. Results are expressed as <i>IFN</i> cDNA copies per 10<sup>5</sup> copies of β-actin cDNA. It is noteworthy that cells from IFNAR1<sup>0/0</sup> expressed lower basal levels of IFN, likely as a consequence of a disrupted positive feed-back loop for IFN expression in these mice.</p
Hepatocytes respond to IFN-α but not to IFN-λ.
<p><b>A:</b> Fluorescence microscopy images showing the IFN response (Mx1, green) in hepatocytes (HNF4, red), in liver sections from Mx-IFNLR1<sup>0/0</sup> and Mx-IFNAR1<sup>0/0</sup> mice infected for 48 hours with influenza A virus strain TURH. Scale bar: 15 µm. <b>B:</b> Fluorescence microscopy images showing the IFN-λ response (Mx1, green) in cholangiocytes (cytokeratin, red) in liver sections from Mx-IFNAR1<sup>0/0</sup> mice, mock-infected or infected for 48 hours with the TURH strain of influenza A virus or mock-infected. Scale bar: 15 µm. Results are representative of two independent experiments.</p