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The result of the antiviral assay presented in figure 2A of U. Müller et al. [Science 264, 1918 (1994)] is consistent with our present results. To investigate further the extent to which the loss of IFNAR1 affects IFN-γ-induced gene expression, we examined the mRNA induction kinetics for IRF-1 and 2′.5′OAS genes by IFN-γ in the WT and IFNAR1-null MEFs. The induction of IRF-1 mRNA by IFN-γ was six times lower in the mutant MEFs than in the WT MEFs. We also examined the induction of 2′,5′OAS mRNA, which is dependent on ISGF3, in the WT and IFNAR1-null MEFs. The induction of 2′,5′OAS mRNA by IFN-γ remained undetectable in IFNAR1-null MEFs throughout the time course tested (19).
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IFNAR1-deficient and IFNGR1-deficient mice were purchased from B&K Universal Group (North Humberside, UK). MEFs and splenocytes were prepared according to standard methods [A. Takaoka et al., EMBO J. 18, 2480 (1999)]. Recombinant murine IFN-γ was purchased from Genzyme/Techne.
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Takaoka, A.1
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0343802751
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note
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Flow cytometry and immunoblot analyses revealed that the expression levels of IFNGR1 and IFNGR2 in these cells are essentially the same as in the WT MEFs. Immunoblot analysis showed that nearly the same amount of endogenous Stat1 protein was detected in the WT and IFNAR1-null MEFs.
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19
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0343367074
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Supplemental Web material is available at Science Online at www.sciencemag.org/feature/data/1049334.shl.
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20
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0343802749
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unpublished results
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M. Sato, unpublished results.
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Detergent-free caveolae fractions were prepared as described [E. J. Smart, Y. S. Ying, C. Mineo, R. G. Anderson, Proc. Natl. Acad. Sci. U.S.A. 92, 10104 (1995); C. Mineo, G. L. James, E. J. Smart, R. G. Anderson, J. Biol. Chem. 271, 11930 (1996)]. The reversibility of the effects of filipin treatment was investigated as described [J. E. Schnitzer, P. Oh, E. Pinney, J. Allard, J. Cell. Biol. 127, 1217 (1994); R. Xavier, T. Brennan, Q. Li, C. McCormack, B. Seed, Immunity 8, 723 (1998)]. Briefly, MEFs treated with filipin (Sigma) for 10 min were washed, incubated for 60 min in Dulbecco's modified Eagle's medium containing 20% fetal calf serum, and then stimulated with IFN-γ.
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Detergent-free caveolae fractions were prepared as described [E. J. Smart, Y. S. Ying, C. Mineo, R. G. Anderson, Proc. Natl. Acad. Sci. U.S.A. 92, 10104 (1995); C. Mineo, G. L. James, E. J. Smart, R. G. Anderson, J. Biol. Chem. 271, 11930 (1996)]. The reversibility of the effects of filipin treatment was investigated as described [J. E. Schnitzer, P. Oh, E. Pinney, J. Allard, J. Cell. Biol. 127, 1217 (1994); R. Xavier, T. Brennan, Q. Li, C. McCormack, B. Seed, Immunity 8, 723 (1998)]. Briefly, MEFs treated with filipin (Sigma) for 10 min were washed, incubated for 60 min in Dulbecco's modified Eagle's medium containing 20% fetal calf serum, and then stimulated with IFN-γ.
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Detergent-free caveolae fractions were prepared as described [E. J. Smart, Y. S. Ying, C. Mineo, R. G. Anderson, Proc. Natl. Acad. Sci. U.S.A. 92, 10104 (1995); C. Mineo, G. L. James, E. J. Smart, R. G. Anderson, J. Biol. Chem. 271, 11930 (1996)]. The reversibility of the effects of filipin treatment was investigated as described [J. E. Schnitzer, P. Oh, E. Pinney, J. Allard, J. Cell. Biol. 127, 1217 (1994); R. Xavier, T. Brennan, Q. Li, C. McCormack, B. Seed, Immunity 8, 723 (1998)]. Briefly, MEFs treated with filipin (Sigma) for 10 min were washed, incubated for 60 min in Dulbecco's modified Eagle's medium containing 20% fetal calf serum, and then stimulated with IFN-γ.
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Detergent-free caveolae fractions were prepared as described [E. J. Smart, Y. S. Ying, C. Mineo, R. G. Anderson, Proc. Natl. Acad. Sci. U.S.A. 92, 10104 (1995); C. Mineo, G. L. James, E. J. Smart, R. G. Anderson, J. Biol. Chem. 271, 11930 (1996)]. The reversibility of the effects of filipin treatment was investigated as described [J. E. Schnitzer, P. Oh, E. Pinney, J. Allard, J. Cell. Biol. 127, 1217 (1994); R. Xavier, T. Brennan, Q. Li, C. McCormack, B. Seed, Immunity 8, 723 (1998)]. Briefly, MEFs treated with filipin (Sigma) for 10 min were washed, incubated for 60 min in Dulbecco's modified Eagle's medium containing 20% fetal calf serum, and then stimulated with IFN-γ.
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0033522422
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Preparation of whole-cell extracts, binding reactions, and gel electrophoresis were done as described [A. Takaoka et al., EMBO J. 18, 2480 (1999)]. Unless otherwise noted, cells were treated with IFN-γ (250 U/ml) for 30 min. Equal amounts of proteins from whole-cell extracts were loaded onto each lane. Experiments were done in triplicate and were performed separately with at least two independent clones of MEFs derived from the same littermates of the WT or mutant mice.
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EMBO J.
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Takaoka, A.1
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7O1) (New England BioLabs); anti-IFNAR1, anti-IFNGR1, and anti-IFNGR2 (Research Diagnostics); and anti-caveolin (Transduction Laboratories).
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EMBO J.
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Takaoka, A.1
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0343367070
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note
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We thank G. Uzé for the murine IFNAR1 cDNA (Gen-Bank accession number M89041); J. Vilcek and E. L. Barsoumian for invaluable advice; M. Asagiri, N. Hata, Y. Kato, and S. H. Kim for technical support; Sumitomo Pharmaceuticals for purified murine IFN-α; and Toray Industries for recombinant murine IFN-β. Animal care was in accordance with institutional guidelines of the University of Tokyo. Supported by grants from the Research for the Future Program (96L00307), Yamanouchi Foundation for Research on Metabolic Disorders, the Japan Society for the Promotion of Science, Advanced Research on Cancer, and the Human Frontier Science Program Organization.
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