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Volumn 284, Issue 5416, 1999, Pages 951-955

Requirement for type 2 NO synthase for IL-12 signaling in innate immunity

Author keywords

[No Author keywords available]

Indexed keywords

ALPHA INTERFERON; BETA INTERFERON; GAMMA INTERFERON; INDUCIBLE NITRIC OXIDE SYNTHASE; INTERLEUKIN 12; NITRIC OXIDE; NITRIC OXIDE SYNTHASE; UNCLASSIFIED DRUG;

EID: 0033532294     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5416.951     Document Type: Article
Times cited : (175)

References (56)
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    • 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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    • 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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    • 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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    • 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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    • -/- 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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