Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-8

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>eactions were performed in the presence of cold UTP and [α-P] UTP (lane 3), or in the presence of single [α-P] UTP (lane 4). An RNA product from the standard RdRp reaction mixture is shown as a control (lane 2). Lane 1, 5'-end labeled 83-nt RNA size marker

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-6

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>oduct synthesized by JEV NS5 using the 83-nt RNA template. Products were resolved on a 5% polyacrylamide sequencing gel (20 × 40 cm) containing 8 M urea. The RNA size markers, 5'-end labeled RNA template (End), and a set of labeled RNA fragments generated by alkaline hydrolysis of the 5'-end labeled RNA template (End/OH), were resolved on the same gel. Arrowhead indicates the internally initiated 81-nt RNA product. (B) The close-up autoradiogram of the same gel shown in (A). (C) Secondary structure of the 83-nt RNA template predicted by the Mfold program. Bent arrow denotes the predicted RNA synthesis initiation site

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-3

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>nce of increasing concentrations of MgClor MnCl(lanes 2–6 and 7–11; 0.5, 1.0, 2.5, 5.0, and 10 mM of MgCland MnCl, respectively). (B) RdRp assays were performed with the 83-nt RNA representing the 3' end of the plus-strand JEV genome, in the absence of metal ions (lane 1) or in the presence of 2.5 mM of the divalent metal ion indicated above the autoradiogram (lanes 2 and 3). RdRp products were denatured and resolved on a medium size (20 × 20 cm) denaturing 5% polyacrylamide gel, and subjected to autoradiography

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-4

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>or absence (-) of JEV 3'(+)UTR RNA (A and C), 3' (-)UTR RNA (B and C), and the 83-nt RNA (D) template. RdRp products were analyzed as in Figure 4 by autoradiography. Arrowheads indicate the template positions visualized by ethidium-bromide staining of the gels

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-2

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>rmed with the poly(A)/(U)template under the indicated conditions. The RdRp activity was measured as in Figure 2 and is presented as the percentage of that observed under each optimal condition. Shown is the mean and standard error from three independent experiments

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-7

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>hown below the structures predicted by the Mfold program. Partial structures representing the lower stem-region are shown. The deleted and added nucleotides are indicated by ρ and boxed, respectively. (B) RNA synthesis directed by the wild-type 83-nt RNA and its derivatives. RdRp assays were performed with the RNA templates indicated above the autoradiogram, and products were resolved a 5% polyacrylamide sequencing gel (20 × 40 cm) containing 8 M urea. Arrowhead indicates the 81-nt internally initiated RNA product. Representative data from three independent experiments are shown

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-1

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>) RNA template in the presence (+) or absence (-) of the primer oligo(U). (B) RdRp assays were performed with the purified wild-type NS5 (GDD) and its mutant NS5(GAD) in the presence (+) or absence (-) of a poly(A) RNA template. Relative RdRp activities (%), which were obtained by comparing the P-UMP incorporation measured by liquid scintillation counting with that obtained for the reaction with the template and primer, 3.0 × 10cpm, are presented

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-5

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>the 3'-terminal region of JEV genome, were left untreated (-) or digested with nuclease S1 (S1), and resolved on a denaturing polyacrylamide gel. Nuclease S1 treatments were performed in 50 mM NaCl (L; low salt) or 500 mM NaCl (H; high salt). Arrowhead indicates the position of the 83-nt RNA template

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase-0

<p><b>Copyright information:</b></p><p>Taken from "Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase"</p><p>http://www.biomedcentral.com/1471-2199/8/59</p><p>BMC Molecular Biology 2007;8():59-59.</p><p>Published online 11 Jul 2007</p><p>PMCID:PMC1934914.</p><p></p>and ion-exchange chromatography using an SP-Sepharose column. (A) Imidazole elution profile of JEV NS5 from Ni-NTA resin. (B) Gel filtration chromatography elution profile of JEV NS5. (C) NaCl-elution profile of JEV NS5 from an SP-Sepharose column. (A-C) Fractions from each purification step were resolved by SDS-12% PAGE and stained with Coomassie brilliant blue. (D) JEV NS5 and its mutant NS5from a peak fraction eluted from an SP-Sepharose column were resolved by SDS-12% PAGE and visualized by silver staining. (E) Western blot analysis of the purified JEV NS5 and NS5using an anti-Hisantibody. The sizes of protein markers are indicated in kilodaltons. Closed and open arrowheads indicate the full-length JEV NS5, and its major cleaved form identified by MALDI-TOF analysis, respectively

DUSP1 suppression increases STAT1 activity and nuclear translocation.

<p>Phosphorylation (A) and nuclear translocation (B) of STAT1 were assessed by cell-based ELISA and confocal microscopy, respectively. (A) Expression of phospho-STAT1 was normalized to that of STAT1 and then to the ratio in LV-cont-infected cells. (B) Red, anti-STAT1; blue, DAPI. Merged image allows assessment of nuclear localization of STAT1; original magnification ×200. All data are representative of at least three independent experiments.</p