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Human full-length and mini TyrRSs, the extra COOH-terminal domain of human TyrRS, E. coli TyrRS, and human mature EMAP II (with a COOH-terminal tag of six histidine residues) were over-expressed in E. coli strain BL 21 (DE 3) (Novagen, Madison, WI) by induction with isopropyl β-D-thiogalactopyranoside for 4 hours. Proteins were purified on a nickel affinity column (His®Bind resin; Novagen) from the supernatant of lysed cells. Endotoxin was removed from the protein solutions by phase separation using Triton X-114 [Y. Aida and M. J. Pabst, J. Immunol. Methods 132, 191 (1990); S. Liu et al., Clin. Biochem. 30, 455 (1997).] and was determined to be <0.01 endotoxin units per milliliter by a Limulus Amebocyte Lysate gel-clot assay (E-Toxate; Sigma, St. Louis, MO).
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Human full-length and mini TyrRSs, the extra COOH-terminal domain of human TyrRS, E. coli TyrRS, and human mature EMAP II (with a COOH-terminal tag of six histidine residues) were over-expressed in E. coli strain BL 21 (DE 3) (Novagen, Madison, WI) by induction with isopropyl β-D-thiogalactopyranoside for 4 hours. Proteins were purified on a nickel affinity column (His®Bind resin; Novagen) from the supernatant of lysed cells. Endotoxin was removed from the protein solutions by phase separation using Triton X-114 [Y. Aida and M. J. Pabst, J. Immunol. Methods 132, 191 (1990); S. Liu et al., Clin. Biochem. 30, 455 (1997).] and was determined to be <0.01 endotoxin units per milliliter by a Limulus Amebocyte Lysate gel-clot assay (E-Toxate; Sigma, St. Louis, MO).
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unpublished observations, [supplementary data can be found at the Science Web site]
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note
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Human MPs and PMNs were prepared from acid citrate dextrose - treated:blood of normal healthy volunteers (8).
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23
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0344434933
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note
-
125I was performed by Research & Diagnostic Antibody (Richmond, CA).
-
-
-
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30
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0025900818
-
-
125I-human mini TyrRS (specific activity of ∼60 Ci/ mmol) in the absence or presence of a 200-fold molar excess of either unlabeled TyrRSs, human mature EMAP II, human recombinant IL-8 (Calbiochem, La Jolla, CA), human recombinant Groα (Biosource International, Camaritlo, CA), or human recombinant NAP-2 (Biosource International). Cells were separated from unbound radioactivity by centrifugation at approximately 8000g for 2 min through 500 μl of a 10% sucrose/phosphate-buffered saline (PBS) cushion. The supernatant was aspirated, and the cell sediment was resuspended by using Ecolite (ICN Biomedicals, Irvine, CA) and analyzed in a scintillation counter.
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Baggiolini, M.5
-
31
-
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0028222920
-
-
125I-human mini TyrRS (specific activity of ∼60 Ci/ mmol) in the absence or presence of a 200-fold molar excess of either unlabeled TyrRSs, human mature EMAP II, human recombinant IL-8 (Calbiochem, La Jolla, CA), human recombinant Groα (Biosource International, Camaritlo, CA), or human recombinant NAP-2 (Biosource International). Cells were separated from unbound radioactivity by centrifugation at approximately 8000g for 2 min through 500 μl of a 10% sucrose/phosphate-buffered saline (PBS) cushion. The supernatant was aspirated, and the cell sediment was resuspended by using Ecolite (ICN Biomedicals, Irvine, CA) and analyzed in a scintillation counter.
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Schraufstätter, I.U.1
Burger, M.2
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Takamori, H.5
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34
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0028280658
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L. Ghibelli, S. Coppola, C. Nosseri, A. Bergamini, S. Beninati, FEBS Lett. 344, 35 (1994).
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Ghibelli, L.1
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Beninati, S.5
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35
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0344866732
-
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note
-
6 cells) were cultured in RPMI 1640 medium without or with heat-treated fetal bovine serum (10%). Cells were washed twice with ice-cold PBS, and resuspended in lysis buffer containing 25 mM Hepes (pH 7.5), 5 mM EDTA, 5 mM dithiothreitol, 0.1% CHAPS detergent, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml aprotinin, 20 μg/ml leupeptin, and 10 μg/ml pepstatin A. The cells were frozen and thawed three times in liquid nitrogen, and then centrifuged for 30 min at 4°C. For examination of proteins in the cell supernatant, 20 ml of spent culture medium (to which the following protease inhibitors were added: 2 mM PMSF, 10 μg/ml aprotinin, 20 μg/ml leupeptin, and 10 μg/ml pepstatin A) was concentrated in a Centriprep-10 (Amicon, Beverly, MA), and the samples were then analyzed on 12.5% SDS-polyacrylamide gels. After transfer onto Immobilon-P transfer membrane (Millipore, Bedford, MA), the membranes were blocked with PBS and 3% BSA, and incubated with rabbit polyclonal antibodies against human full-length TyrRS. Washed membranes were then incubated with a horseradish peroxidase-linked antibody to rabbit immunoglobulin (1:4000 dilution; Amersham Life Science, Arlington Heights, IL) for detection of TyrRS.
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36
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0344434935
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note
-
LDH activities were determined spectrophotometrically with a CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI).
-
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-
40
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0030053586
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L. Ribas de Pouplana, M. Frugier, C. L. Quinn, P. Schimmel, Proc. Natl. Acad. Sci. U.S.A. 93, 166 (1996).
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Ribas De Pouplana, L.1
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42
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0027049163
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-
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Richmond, A.1
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46
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0344434929
-
-
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, Ile; 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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47
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0344866730
-
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note
-
We thank I. U. Schraufstātter for RBL2H3 rat basophilic leukemia cells expressing IL-8 type A or B receptor, and D. J. Loskutoff for human U-937 cells. Supported by NIH grant GM23562 and by a grant from the National Foundation for Cancer Research. K.W. was supported by a JSPS (Japan Society for the Promotion of Science) Postdoctoral Fellowships for Research Abroad.
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