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Volumn 274, Issue 5288, 1996, Pages 787-789

Suppression of TNF-α-induced apoptosis by NF-κB

Author keywords

[No Author keywords available]

Indexed keywords

IMMUNOGLOBULIN ENHANCER BINDING PROTEIN; TUMOR NECROSIS FACTOR ALPHA;

EID: 0029992609     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5288.787     Document Type: Article
Times cited : (2477)

References (36)
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    • IκBαM was generated by digestion of plasmids pCMX-IκBαS32/36A and pCMX-IκBαMutF with Eco Nl and Bst Ell and ligation of the small S32/36A fragment to the large vector/1κBαMutF fragment. The resulting expression plasmid called pCMX-IκBαM, was confirmed by in vitro transcription and translation (TNT, Promega) with the T7 promoter present in pCMX followed by immunoprecipitation with IκBα-specific antibody. pCMX-IκBαS32/36A was constructed by removal of the Bam Hl-Hind III fragment of pBS-IκBαS32/36A and ligation of the fragment into the Bam Hl-Hind III sites in pCMX. pBS-IκBαS32/36A was constructed by site-directed mutagenesis of the plasmid pBS-IκBα and confirmed by sequencing. The construction of pCMX-IκBαMutF has been described (5). pLIκBαMSN was constructed by blunt insertion of the Eco RV fragment from pCMX-IκBαM into the Hpa I site of pLXSN (25).
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    • K. Brown, S. Gerstberger, L. Carlson, G. Franzoso, U. Siebenlist, Science 267, 1485 (1995); E. B. Traenckner et al., EMBO J. 14, 2876 (1995).
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    • 10544253523 scopus 로고    scopus 로고
    • note
    • 4 cells in 3 ml with Polybrene (8 μg/ml) for 8 to 12 hours. Cells were allowed to expand for 48 hours and were then selected for neomycin resistance. Amounts of G418 used in selecting the various cell types were as follows: MEF and HEF cells, 800 μg/ml; T24,400 μg/ml; Jurkat. 1 mg/ml, Immunoblotting was then performed to analyze the expression of IκBαM.
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    • 2-terminus of IκBα (C-15, Santa Cruz Biotech) and diluted 1:1000 in phosphate-buffered saline (PBS) with 0.2% Tween-20 (Sigma) and 5% nonfat milk (Carnation). After washing, the blots were probed with horseradish peroxidase (HRP)-conjugated donkey antiserum to rabbit immunoglobulin G (Amersham) diluted 1:3000 for 2 hours. Bands were visualized by use of the Renaissance chemiluminescence kit (Dupont).
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    • note
    • -32P] adenosine triphosphate (Dupont NEN), TNF-α, IL-1α, phorbol 12-myristate 13-acetate (PMA), and ionomycin A23187 were obtained from Calbiochem.
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    • 10544230339 scopus 로고    scopus 로고
    • note
    • Cytospin analysis was done as described (31). Briefly, cells were treated as described in the text, and a 100-μl sample was loaded into disposable chambers and centrifuged for 1 min onto glass slides in a cytocentrifuge (Shandon) The slides were allowed to dry and then stained with the Leukostat stain kit (Fisher Scientific). Staining was verified by visualization under a light microscope, and the slides were mounted with glass cover slips with the use of Permount mounting media (Fisher Scientific). The population of cells was categorized as either viable, apoptotic, or necrotic when analyzed by staining morphology. Counting was performed blind, meaning that each slide was given a number and the identity of each sample was not known during counting. The counting procedure made use of a light microscope at a magnification that allowed the incorporation of about 100 cells per field. Five fields were counted per slide, and each experimental condition was performed two times.
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    • note
    • 5 per milliliter and treated identically to MEF cells. Quantitation was done with Cell Quest software.
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    • N. Itoh et al., Cell 66, 233 (1991).
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    • Itoh, N.1
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    • L. Naldini et al., Science 272, 263 (1996).
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    • note
    • We are grateful to B. Sha, A. Beg, and D. Baltimore for p50 and RelA knockout fibroblast cell lines and to D Trono for Jurkat cells We thank members of the Verma laboratory for helpful discussions, P. Charon for help in manuscript preparation, and D. Finucane for assistance with flow cytometry. D.J.V. is a graduate student in the Department of Chemistry and Biochemistry at the University of California, San Diego, and is supported by a fellowship from the Chapman Charitable Trust, S.J.M. is a Wellcome Trust Senior Fellow. T.K. is supported by a European Molecular Biology Organization Fellowship. D.R.G.'s laboratory is supported by grants GM52735 from the National Institutes of Health and CB-82 from the American Cancer Society. I.M.V.'s laboratory is supported by grants from the NIH and American Cancer Society. I.M.V. Is an American Cancer Society Professor of Molecular Biology.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.