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Volumn 287, Issue 5454, 2000, Pages 869-873

A subclass of Ras proteins that regulate the degradation of IκB

Author keywords

[No Author keywords available]

Indexed keywords

RAS PROTEIN; TRANSCRIPTION FACTOR;

EID: 0034603021     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5454.869     Document Type: Article
Times cited : (91)

References (34)
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    • note
    • Construction of the phylogenic tree was done with the Phylip software package. The Ras similarity domains (corresponding to amino acids 5 to 164 of H-Ras) were aligned by ClustalW alignment and compared by parsimonious analysis with amino acid similarities and bootstrapping analysis. Although variations of the Ras family dendrogram were generated, κB-Ras1 and κB-Ras2 remained grouped with Ras/ Ral/Rap proteins.
  • 19
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    • note
    • Nucleotide binding to κB-Ras1 was performed by slot-blot analysis with bacterially expressed κB-Ras1 with a COOH-terminal His tag. κB-Ras1 was purified on nickel-agarose gel and dialyzed extensively in a buffer containing 1 mM EDTA before the nucleotide-binding assay (32).
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    • unpublished observations
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    • note
    • 6 for Jurkat cells) and grown for 24 hours before transfection with 1 μg of DNA with the Fugene6 method (Boehringer Mannheim). After -24 hours, selected culture cells were induced with TNF-α (10 ng/ml) or recombinant human IL-1β (10 ng/ml) or with phorbol 12-myristate 13-acetate (250 ng/ml) or ionomycin (10 ng/ml). All cell cultures were harvested 4 hours later, and luciferase activity was measured (Promega luciferase assay kit). Transfection control was measured with a renila luciferase vector with a thymidine kinase promoter or by protein concentration.
  • 29
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    • note
    • Cells were harvested and blotted as previously described (25) with membranes probed with antibody to IκBα or IκBβ (C21 and C20; Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal rabbit antibodies, or monoclonal mouse antibody to HA (12CA5). Immunoprecipitations were performed on transfected COS or 293 cells (3.5- and 10-cm plates, respectively), as previously described, with antibody to HA.
  • 33
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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, 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.
  • 34
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    • note
    • This work was supported by the Howard Hughes Medical Institute (HHMI) and NIH (S.G.) and by the National Creative Research Initiatives Program of the Korean Ministry of Science and Technology (J.W.L). C.F. was supported by Fonds Formation Chercheurs et Aide Recherche and HHMI, R.E.V. by Deutsche Forschungsgemeinschaft, and H.Z. by the Leukemia Society of America. We thank I. Douglas for technical help, P. J. Novick for the purified Sec4 protein, D. S. Na for helpful advice, and D. G. Schatz, D. Sengupta, H. J. Kim, and M. Solomon for careful reading of the manuscript.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.