Lentivirus vector preparation and virus production were as described previously 39. HEK293 and HEK293-TLR3 cells were transfected with the luciferase reporter gene plasmids
as described previously 7 and co-transfected with the various expression vectors using Lipofectamine 2000 (Invitrogen). After 24 h, cells were stimulated selleck compound with stimulated with poly(I:C) as indicated. Thereafter, cell lysates were prepared and reporter gene activity was measured using the Dual Luciferase Assay system (Promega) as described previously 40. Data were expressed as the mean fold induction±SD relative to control levels, for a representative experiment from a minimum of three separate experiments, each performed in triplicate. HEK293 or HEK293-TLR3 cells were transfected using Lipofectamine 2000 (Invitrogen) with the indicated plasmids. Twenty-four hours later,
cells were stimulated and lysed as described previously 40. The immune complexes were precipitated, washed, eluted by the addition of sample buffer followed by SDS-PAGE and immunoblotting using the indicated antibodies. BMDM were stimulated with the indicated ligands. After 4 and 16 h, the cell-free supernatants were removed and analysed for IFN-β release according to the manufacturer’s (PML) instructions. IL-6, TNF-α and CCL5 cytokine release were measured as indicated by the manufacturer (Peprotech). Cells were stimulated with ligand as described and lysates were subjected to SDS-PAGE followed by immunoblot analysis learn more with an anti-IRF7 (Santa Cruz), anti-phospho-IRF7 (a generous gift from Professor John Hiscott) anti-IRF3 (Santa Cruz) and anti-phospho-IRF3 antibodies (Cell Signalling). HEK293-TLR3 cells expressing YFP-tagged IRF3 or IRF7 proteins were stimulated with poly(I:C) and at appropriate time points, cells were rinsed with PBS and fixed at RT for
5 min with 2% formaldehyde solution. Cells were counterstained using DAPI nuclear stain (Sigma). Fluorescence was examined using an Olympus IX81 fluorescent microscope (Olympus, Germany). Statistical analysis was carried out using the unpaired Student’s t-test using SigmaPlot 2001 programme. p-Values of less than or equal to 0.05 were considered to indicate a statistically significant difference where * indicated Cyclin-dependent kinase 3 p<0.05 and ** indicates p<0.005. The authors thank Professor Paul Moynagh for critical evaluation of the manuscript. The authors and their work were supported by the Health Research Board of Ireland (RP/2006/293 to S. M.) and Science Foundation Ireland (RP/2008/11 to S. M.). Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. "
“Citation Manaster I, Mandelboim O. The unique properties of uterine NK cells.