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note
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6 L. major promastigotes (strain MHOM/IL/81/FE/BNI) as described (9).
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Recombinant mouse IL-12 (250 to 300 ng per injection; Genetics Institute, Cambridge, MA) or phosphate-buffered saline (PBS) was injected intraperitoneally (i.p.) at 24 and 4 hours before infection. L-NIL (2.5 mg per injection; Searle/Monsanto, St. Louis, MO) or PBS was injected i.p. twice daily, starting 48 to 72 hours before infection.
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0344710029
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-
note
-
IL-12Rβ1 and the IL-12Rβ2 competitors were constructed by the linker primer technique from the original cDNAs and were used with the following primers: IL-12Rβ1 sense, 5′-CGG GGG TCC TGA CGC AAT ACG-3′[base pair (bp) position 1545 → 1565 of the mouse IL-12Rβ1 sequence]; IL-12Rβ1 antisense, 5′-CTC CCG GCA TCT CGA CCA CCA G-3′ (bp position 1976 → 1997); IL-12Rβ2 sense, 5′-CTG CGA GAT CTG AGA CCG TTT ACA-3′ (bp position 1027 → 1050 of the mouse IL-12Rβ2 sequence); IL-12Rβ2 antisense, 5′-GGG GGA TCC GCA GCC AGT G-3′ (bp position 1597 → 1615). The sequences of the linker primers were 5′-CTC GAC CAG CAG GCC CTG TTT AAG CCA ATG T-3′ (IL-12Rβ1) and 5́-CCG CAG CCA GTG TCC GAC TTT GCA GAG ACC T-3′ (IL-12Rβ2). The annealing temperatures were 58°C and the number of polymerase chain reaction (PCR) cycles was 35.
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25
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0345572020
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note
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+ cells by complement-mediated cell lysis with rat antibodies to CD4 and CD8 (gift of T. Winkler, Erlangen, Germany) and by incubation with rat antibody to CD3 (clone KT3, Dianova, Hamburg, Germany), followed by anti-rat immunoglobulin (Ig) Dynabeads (Dynal, Hamburg, Germany).
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30
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0345140418
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note
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+ cells).
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31
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0345140417
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note
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+ cells) before stimulation with rmlL-12 (0.1 to 1 ng/ml) or mouse IFN-α/β (100 to 200 U/ml).
-
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33
-
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0344710026
-
-
note
-
The NK cell clone KY-1 (derived from p53-deficient C57BL/6 mice) shows stable proliferation in the presence of IL-2. Cells were expanded in IL-2 (50 to 400 U/ml), washed, and treated as indicated. Stimulation with IL-12 (0.1 to 1 ng/ml) or mouse IFN-α/β (200 U/ml; gift of I. Gresser, CNRS, Villejuif, France) for ≤24 hours did not require the routine addition of fresh IL-2.
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34
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0344710024
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See Web Fig. 1 at
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See Web Fig. 1 at www.sciencemag.org/feature/data/ 986543.shl.
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35
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0344710023
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note
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+ <1%), and stimulated with rmlL-12 (0.1 to 1 ng/ml) or mouse IFN-α/β (100 to 500 U/ml).
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36
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0345572019
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note
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After stimulation, KY-1 cells or IL-2-expanded splenic NK cells were washed twice with ice-cold PBS (plus 100 μM sodium orthovanadate) and lysed in 20 mM tris buffer (pH 8.0) containing 150 mM NaCl; 1% Triton X-100; 0.5% NP-40; 1 mM each of EDTA, EGTA, sodium 'orthovanadate, sodium pyrophosphate, sodium fluoride, and PMSF; 0.1 mM sodium molybdate; and pepstatin A, aprotinin, chymostatin, and leupeptin (5 μg/ml each). Protein lysates were immunoprecipitated with 1 μg of affinity-purified rabbit anti-mouse Stat4 IgG (C-20, Santa Cruz Biotechnology, Santa Cruz, CA) in the absence or presence of the respective Stat4 blocking peptide (5 μg/ml) using protein A/G-Plus-agarose (Santa Cruz Biotechnology); separated by 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE); and transferred to nitrocellulose. Immunoblotting (IB) of tyrosine phosphorylated Stat4 was performed with anti-phosphotyrosine mouse IgG [PY99, Santa Cruz Biotechnology; 1 μg/ml in dilution buffer (tris-buffered saline with 5% nonfat dry milk and 0.05% Tween 20)], followed by incubation with peroxidase-conjugated goat anti-mouse IgG (Dianova; 160 ng/ml in dilution buffer) and detection with ECL Plus (Amersham Pharmacia Biotech). Blots were reprobed with anti-Stat.4 IgG (200 ng/ml in dilution buffer).
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0344278011
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See Web Fig. 2 at
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See Web Fig. 2 at www.sciencemag.org/feature/data/ 986543.shl.
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C. M. Bacon et al., J. Exp. Med. 181, 399 (1995); S. J. Szabo, N. G. Jacobson, A. S. Dighe, U. Gubler, K. M. Murphy, Immunity 2, 665 (1995); J. J. O'Shea, ibid. 7, 1 (1997).
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C. M. Bacon et al., J. Exp. Med. 181, 399 (1995); S. J. Szabo, N. G. Jacobson, A. S. Dighe, U. Gubler, K. M. Murphy, Immunity 2, 665 (1995); J. J. O'Shea, ibid. 7, 1 (1997).
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0345140415
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note
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KY-1 cells, primary NK cells, or T cells were lysed (27) and immunoprecipitated with rabbit anti-jak1 IgG (HR-78S, Santa Cruz Biotechnology; 2 μg/ml), rabbit anti-Jak2 IgG (HR-758, Santa Cruz Biotechnology; 2 μg/ml), or rabbit anti-Tyk2 (C-15, specific for amino acids 1173 through 1187 [O. Colamonici et al., Mol. Cell. Biol. 14, 8133 (1994)]; 1:200) in the absence or presence of the respective blocking peptides (10 μg/ ml) using protein A - Sepharose. They were then separated by 7.5% SDS-PAGE and transferred to nitrocellulose. Immunoblotting of tyrosine-phosphorylated Jak1, Jak2, or Tyk2 was performed with antiphosphotyrosine mouse IgG (PY99, 1 μg/ml; Santa Cruz Biotechnology) as described (27). After stripping, blots were reprobed with antibody to Jak1 (800 ng/ml), Jak2 (600 ng/ml), or Tyk2 [C-15 combined with C-20 (Santa Cruz Biotechnology), 600 ng/ml].
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48
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0344710020
-
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note
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32P)ATP (>3000 Ci/mmol) and incubated for 30 min at room temperature. After several washes in tris buffer (pH 7.5) containing 0.1% Triton X-100 and 100 μM orthovanadate and a final wash in Triton-free tris buffer (pH 6.8), the immunoprecipitates were eluted in Laemmli-loading buffer, fractionated on 7.5% SDS-PAGE, and transferred to reinforced nitrocellulose (Schleicher & Schuell, Dassel, Germany) for autoradiography. In some cases, the protein bead immunocomplexes were treated with DETA/NO or DETA (1 to 100 μM in kinase buffer) for 60 min before the in vitro kinase reaction.
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49
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0344710019
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See Web Fig. 3 at
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See Web Fig. 3 at www.sciencemag.org/feature/data/ 986543.shl.
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See Web Fig. 4 at
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See Web Fig. 4 at www.sciencemag.org/feature/data/ 986543.shl.
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0344278007
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note
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Cell lysates were prepared in 40 mM tris buffer (pH 8.0) containing 200 μM phenylmethylsulfonyl fluoride and aprotinin, chymostatin, pepstatin A, and leupeptin (5 μg/ml each), then separated on a 7.5% SDS-PACE followed by protein immunoblotting with a rabbit anti-mouse NOS2 IgG (9) using the ECL Plus detection system (Amersham Pharmacia Biotech, Freiburg, Germany).
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56
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0344278008
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-/- mice; C. Brooks, A. Ding, M. Müller, and E. Parganas for advice; D. Raulet and R. E. Vance for discussions; K. Schröppel for help with the electronic data files; and E. Lorenz and N. Donhauser for their outstanding technical assistance.
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