Abstract:
:We generated and characterized novel antibodies specific for a cleavage site of human caspase-8/FLICE and its substrate, FLICE-like inhibitory protein (FLIP). The synthetic peptides used as immunogens were CQGDNYQKGIPVETD (#791) and VSEGQLEDSSLLEVD (#1342), which corresponded to cleaved regions of N-terminal fragments of caspase-8 and FLIP generated by active caspase-8, respectively. Each antibody purified from rabbit antiserum reacted specifically with the immunogen but not with the peptide corresponding to the unproteolyzed form, as assessed by ELISA. In vitro cleavage of GST-FLIP by active caspase-8 generated an N-terminal fragment (GST-p43) and a C-terminal one (p12). Consistent with other in vivo data, the FLIP cleavage site follows the Asp residue, LEVD(376)GPAMKNVEF, identified on N-terminal sequencing of the p12 fragment. #1342-antibody (#1342-Ab) recognized the GST-p43 fragment but not the uncleaved protein, thus confirming its specificity. When the antibodies were used for immunoblotting, flow cytometry, and confocal laser microscopy, the proteolysis of caspase-8 and FLIP, and the subcellular localization of their digests could be monitored in apoptotic U937 cells. Interestingly, a significant increase in the percentage of cells exhibiting caspase-8 and FLIP cleavage was observed upon Fas stimulation in interferon-gamma-treated U937 cells, in which the susceptibility to Fas is extremely enhanced. In contrast, U937 cells treated with vitamin D(3) or all-trans retinoic acid showed Fas-resistance, and caspase-8 processing and FLIP cleavage were strongly inhibited. In conclusion, we established a system based on the cleavage site-directed antibodies to monitor the dynamics of caspase-8 processing and activation during apoptosis. Using this system, we found that Fas-susceptibility changes during U937 differentiation occur upstream of caspase-8 processing/activation.
journal_name
J Biochemjournal_title
Journal of biochemistryauthors
Niikura Y,Nonaka T,Imajoh-Ohmi Sdoi
10.1093/oxfordjournals.jbchem.a003198keywords:
subject
Has Abstractpub_date
2002-07-01 00:00:00pages
53-62issue
1eissn
0021-924Xissn
1756-2651journal_volume
132pub_type
杂志文章abstract::Three forms (termed I, II, and III) of ribonuclease H (RNase H) [EC 3.1.4.34] activity are present in rat liver cytosol. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at 0 M, 0.25 M, and 0.5 M KCl in phosphocellulose chromatography, and were further purified by using blue Sephar...
journal_title:Journal of biochemistry
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pub_type: 杂志文章
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pub_type: 杂志文章
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更新日期:2001-11-01 00:00:00
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pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a021478
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pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a122150
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pub_type: 杂志文章
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pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a123797
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a135300
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a131845
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a133070
更新日期:1980-10-01 00:00:00
abstract::Rat and mouse cDNAs for Zn-alpha 2-glycoprotein (Zn alpha 2gp) were isolated from liver libraries (lambda gt11) and compared with the human one. The lengths of cDNA inserts analyzed were 1,233 and 1,273 nucleotides for rat and mouse, respectively. The deduced amino acid sequences suggested that rat and mouse Zn alpha ...
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pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a124579
更新日期:1994-09-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a133081
更新日期:1980-10-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a134438
更新日期:1983-09-01 00:00:00
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journal_title:Journal of biochemistry
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doi:10.1093/jb/mvq156
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:
更新日期:1980-07-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:
更新日期:1979-05-01 00:00:00
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pub_type: 杂志文章
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pub_type: 杂志文章
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journal_title:Journal of biochemistry
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:1984-03-01 00:00:00
abstract::We investigated whether transforming growth factor (TGF)-β1 promoted epithelial-mesenchymal transition (EMT) and migration of human oral squamous cell carcinoma (hOSCC) cells. Among 6 hOSCC cell lines investigated, Smad2 phosphorylation and TGF-β target genes expression were most clearly upregulated following TGF-β1 s...
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