Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H||IP, WB, ICC||M||Purified||Monoclonal Antibody|
|Description||Anti-p80 Ku Antibody, clone GE2.9.5|
|Presentation||0.1M Tris-glycine, pH 7.4, 0.15M NaCl, 0.05% sodium azide|
|Application||Anti-p80 Ku Antibody, clone GE2.9.5 is a high quality Mouse Monoclonal Antibody for the detection of p80 Ku & has been validated in IP, WB, ICC.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||2 years at -20°C|
|Material Size||200 µg|
|Anti-p80 Ku, clone GE2.9.5 - 15391||15391|
|Anti-p80 Ku, clone GE2.9.5 - 18430||18430|
|Anti-p80 Ku, clone GE2.9.5 - 25201||25201|
|Reference overview||Application||Pub Med ID|
|A gene expression signature identifying transient DNMT1 depletion as a causal factor of cancer-germline gene activation in melanoma.|
Cannuyer, J; Van Tongelen, A; Loriot, A; De Smet, C
Clinical epigenetics 7 114 2015
Many human tumors show aberrant activation of a group of germline-specific genes, termed cancer-germline (CG) genes, several of which appear to exert oncogenic functions. Although activation of CG genes in tumors has been linked to promoter DNA demethylation, the mechanisms underlying this epigenetic alteration remain unclear. Two main processes have been proposed: awaking of a gametogenic program directing demethylation of target DNA sequences via specific regulators, or general deficiency of DNA methylation activities resulting from mis-targeting or down-regulation of the DNMT1 methyltransferase.By the analysis of transcriptomic data, we searched to identify gene expression changes associated with CG gene activation in melanoma cells. We found no evidence linking CG gene activation with differential expression of gametogenic regulators. Instead, CG gene activation correlated with decreased expression of a set of mitosis/division-related genes (ICCG genes). Interestingly, a similar gene expression signature was previously associated with depletion of DNMT1. Consistently, analysis of a large set of melanoma tissues revealed that DNMT1 expression levels were often lower in samples showing activation of multiple CG genes. Moreover, by using immortalized melanocytes and fibroblasts carrying an inducible anti-DNMT1 small hairpin RNA (shRNA), we demonstrate that transient depletion of DNMT1 can lead to long-term activation of CG genes and repression of ICCG genes at the same time. For one of the ICCG genes (CDCA7L), we found that its down-regulation in melanoma cells was associated with deposition of repressive chromatin marks, including H3K27me3.Together, our observations point towards transient DNMT1 depletion as a causal factor of CG gene activation in vivo in melanoma.
|Subcellular proteomics reveals a role for nucleo-cytoplasmic trafficking at the DNA replication origin activation checkpoint.|
Mulvey, CM; Tudzarova, S; Crawford, M; Williams, GH; Stoeber, K; Godovac-Zimmermann, J
Journal of proteome research 12 1436-53 2013
Depletion of DNA replication initiation factors such as CDC7 kinase triggers the origin activation checkpoint in healthy cells and leads to a protective cell cycle arrest at the G1 phase of the mitotic cell division cycle. This protective mechanism is thought to be defective in cancer cells. To investigate how this checkpoint is activated and maintained in healthy cells, we conducted a quantitative SILAC analysis of the nuclear- and cytoplasmic-enriched compartments of CDC7-depleted fibroblasts and compared them to a total cell lysate preparation. Substantial changes in total abundance and/or subcellular location were detected for 124 proteins, including many essential proteins associated with DNA replication/cell cycle. Similar changes in protein abundance and subcellular distribution were observed for various metabolic processes, including oxidative stress, iron metabolism, protein translation and the tricarboxylic acid cycle. This is accompanied by reduced abundance of two karyopherin proteins, suggestive of reduced nuclear import. We propose that altered nucleo-cytoplasmic trafficking plays a key role in the regulation of cell cycle arrest. The results increase understanding of the mechanisms underlying maintenance of the DNA replication origin activation checkpoint and are consistent with our proposal that cell cycle arrest is an actively maintained process that appears to be distributed over various subcellular locations.
|DNA-dependent ATPase from HeLa cells is related to human Ku autoantigen.|
Cao, Q P, et al.
Biochemistry, 33: 8548-57 (1994) 1994
A 150-kDa DNA-dependent ATPase composed of 83/68-kDa subunits was previously reported to cofractionate with a 21S complex of enzymes for DNA synthesis from HeLa cells (Vishwanatha, J. K., & Baril, E. F. (1990) Biochemistry 29, 8753-8759). The DNA-dependent ATPase was purified to electrophoretic homogeneity from a HeLa cell homogenate by a modified procedure that involves subcellular fractionation, poly(ethylene-glycol) precipitation of the combined nuclear extract/cytosol, and chromatography on Q-Sepharose and native and denatured DNA/celluloses followed by Mono-S fast protein liquid chromatography. The purified enzyme showed equimolar amounts of 83- and 68-kDa polypeptides following polyacrylamide gel electrophoresis under denaturing conditions. Sequence analysis of peptide fragments derived from the separated 83- and 68-kDa polypeptides showed 90-100% homology with the corresponding 80- and 70-kDa subunits of human Ku protein. Immunoblot analysis of the ATPase during the course of its purification and immunoprecipitation with antibodies to the 80- and 70-kDa subunits of human Ku protein confirmed the relationship of the 83- and 68-kDa polypeptides of the human DNA-dependent ATPase to the subunits of human Ku protein. Both the 83- and 68-kDa polypeptides are phosphorylated by a DNA-dependent protein kinase that cofractionates with the ATPase. The DNA-dependent ATPase activity is up regulated by phosphorylation.
|Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers.|
Wang, J, et al.
J. Cell. Sci., 107 ( Pt 11): 3223-33 (1994) 1994
The Ku autoantigen is a heterodimer of 70 kDa (p70) and -80 kDa (p80) subunits that is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK). The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates Sp-1, Oct-1, p53 and RNA polymerase II in vitro, but the precise cellular role of DNA-PK remains unclear. In the present studies, the assembly of p70/p80 heterodimers and the interaction of Ku with DNA was investigated using recombinant vaccinia viruses directing the synthesis of human p70 (p70-vacc) and p80 (p80-vacc), and monoclonal antibodies (mAbs). Expression of human Ku antigens in rabbit kidney (RK13) cells could be demonstrated by immunofluorescent staining because this cell line contains little endogenous Ku. A novel mAb designated 162 stained the nuclei of RK13 cells coinfected with p70-vacc and p80-vacc, but not cells that were infected with either virus alone, suggesting that it recognized the p70/p80 heterodimer but not monomeric p70 or p80. In agreement with the immunofluorescence data, 162 immunoprecipitated both p70 and p80 from extracts of coinfected cells, but did not immunoprecipitate either subunit by itself from extracts of cells infected with p70-vacc or p80-vacc, respectively. Conversely, the binding of 162 to Ku isolated from human K562 cells stabilized the p70/p80 heterodimer under conditions that normally dissociate p70 from p80. The nuclei of cells infected with p70-vacc alone could be stained with mAb N3H10 (anti-p70) and cells infected with p80-vacc alone could be stained with mAb 111 (anti-p80), indicating that the formation of p70/p80 heterodimers was not required for nuclear transport. Finally, free recombinant and cellular p70 both bound to DNA efficiently in vitro, suggesting that free p70, like the p70/p80 heterodimer, serves as a DNA-binding factor. Moreover, free human p70 could be released from the nuclei of p70-vacc-infected RK13 cells by deoxyribonuclease I treatment, suggesting that it was associated with chromatin in vivo. The nuclear transport of free p70 and the association of free p70 with chromatin in vivo raise the possibility that newly synthesized cellular p70 might undergo nuclear transport and DNA-binding prior to dimerization with p80 or assembly with p350.
|Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen.|
Tuteja, N, et al.
EMBO J., 13: 4991-5001 (1994) 1994
Human DNA helicase II (HDH II) is a novel ATP-dependent DNA unwinding enzyme, purified to apparent homogeneity from HeLa cells, which (i) unwinds exclusively DNA duplexes, (ii) prefers partially unwound substrates and (iii) proceeds in the 3' to 5' direction on the bound strand. HDH II is a heterodimer of 72 and 87 kDa polypeptides. It shows single-stranded DNA-dependent ATPase activity, as well as double-stranded DNA binding capacity. All these activities comigrate in gel filtration and glycerol gradients, giving a sedimentation coefficient of 7.4S and a Stokes radius of approximately 46 A, corresponding to a native molecular weight of 158 kDa. The antibodies raised in rabbit against either polypeptide can remove from the solution all the activities of HDH II. Photoaffinity labelling with [alpha-32P]ATP labelled both polypeptides. Microsequencing of the separate polypeptides of HDH II and cross-reaction with specific antibodies showed that this enzyme is identical to Ku, an autoantigen recognized by the sera of scleroderma and lupus erythematosus patients, which binds specifically to duplex DNA ends and is regulator of a DNA-dependent protein kinase. Recombinant HDH II/Ku protein expressed in and purified from Escherichia coli cells showed DNA binding and helicase activities indistinguishable from those of the isolated protein. The exclusively nuclear location of HDH II/Ku antigen, its highly specific affinity for double-stranded DNA, its abundance and its newly demonstrated ability to unwind exclusively DNA duplexes, point to an additional, if still unclear, role for this molecule in DNA metabolism.
|Antigenic determinants of the 70-kDa subunit of the Ku autoantigen.|
Abu-Elheiga, L and Yaneva, M
Clin. Immunol. Immunopathol., 64: 145-52 (1992) 1992