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2, and 5 mM dithiothreitol (DTT)]. Cleavage of the GST fusion protein with thrombin (∼1 U per milligram of substrate; Novagen) was done overnight. at room temperature, Eluted STAT-4 N-domain (in cleavage buffer) was concentrated to 20 mg/ml with centricon (Amicon) and used for crystallization.
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The polymerization status was determined by dynamic light scattering at a protein concentration of 1 mg/ml using a Protein Solutions dp801 instrument. [U. Vinkemeier and I. Moarefi, unpublished observations].
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6844223705
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note
-
2, 50 mM KCI, and 5 mM DTT. In vitro phosphorylation was done as described (9).
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33
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6844225712
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note
-
Gel-shift experiments and determination of tetramer stability were done as described (9) with an oligonucleotide containing two copies of the STAT recognition element from the c-fos gene (26) spaced by 10 base pairs (5′-GCCAGTCAGTTCCCGTCAATGCATCAGGTTCCCGTCAATGCAT-3′, binding sites underlined). Both protein preparations (Tyr-phosphorylated wild-type STAT-1α and W37A mutant) were titrated in gel-shift experiments with an oligonucleotide containing a single M67 (26) site (5′-GCCGATTTCCCGTAAATCAT-3′) to assure similar loading of active protein.
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34
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6844230888
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note
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Transient transfections were done on six-well plates with 50% confluent U3A cells using the calcium phosphate method as instructed by the manufacturer (Stratagene) with the following modifications. Transfection reactions contained 4.5 μg per well of either wild-type STAT-1α or the W37A mutant in plasmid pcDNA3 (Invitrogen), 4 μg of luciferase reporter plasmid pLuc (C. M. Horvath), and 0.4 μg of β-galactosidase reporter plasmid (Stratagene). The luciferase reporter contained in its Bam HI site as an enhancer element two tandemly arranged weak STAT-1 binding sites (5′-GATCAGTTCCCGTCAATCATGATCCAGTTCCCGTCAATGATCCCCGGGATC-3′) from the human c-fos promoter. Thirty-six hours after transfection, cells were treated with interferon-γ (5 ng/ml, Amgen) for 10 hours or left untreated. Luciferase assays (Promega) and β-galactosidase assays (Stratagene) were done according to the manufacturer's protocol. Protein expression and Tyr-phosphorylation were checked in gel-shift experiments with whole-cell extracts for both wild-type and mutant protein and were comparable. All results shown are luciferase activities normalized to β-galactosictase activity.
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6844263118
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note
-
Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lle; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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40
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42
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6844243223
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note
-
We thank H. Viguet and M. M. Allen for expert technical support, X. Zhu and C. M. Horvath for reagents, and the staff at BNL, especially C. Ogata, for advice in collecting MAD data. U.V. expresses special thanks to C. Harrison and F. Sicheri for valuable and generous help with DENZO and X-PLOR, and to D. E. Drake for assistance with figures. Supported by NIH grants AI34420 and AI32489 to J.E.D.
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