Research Project
Grant-in-Aid for Scientific Research (B)
We have tried to identify physiological substrates of ubiquitin ligase by establishment of comprehensive-analytical method for detection of difference between before and after genetic engineering in knock-out mice.First, we purified ubiquitinated protein specifically by anti-ubiquitin antibody. Ubiquitin is an abundant protein and exist as free form (not conjugated or binding form in mammalian cells. By affinity-chromatography using a conventional antibody, we recovered a free-ubiqitin mainly, and could not collect ubiquitin conjugated proteins. For improvement of the efficiency of collection, we assesed many kinds of antibodies and finally decided to use the FK2 monoclonal antibody, which binds only substrate-conjugated ubiqitin specifically. We succeeded in concentrating ubiquitinated proteins by affinity-chromatography using FK2 binding column.For comparison of proteins between knock-out mice and wild type mice, we have to choose the most adequate tissue and the most effective method for cell-extraction. We identified the tissue which expressed F-box protein we were interested in by in-situ hybridization.Based on these basic informations, we prepared Fbw7-deficient-embryonic fibroblasts and also wild-type fibroblast, and extracted whole cell lysate from each samples. Cell lysate loaded on column packed FK2-antibody conjugated beads for concentration of ubiquitinated proteins. We plan to compare the difference of the ubiquitinated proteins between Fbw7-deficient and wild-type embryonic fibroblasts.
All 2006 2005 2004
All Journal Article (69 results)
EMBO J. 25・5
Pages: 1126-36
Arterioscler. Thromb. Vasc. Biol. 26・4
Pages: 780-6
Oncogene 25・12
Pages: 1786-98
Arterioscler.Thromb.Vase.Biol. 26
Pages: 780-786
Oncogene 25
Pages: 1786-1798
EMBO J. 25
Pages: 1126-1136
Cancer Res. (発表予定)
FASEB J. (発表予定)
EMBO J. (発表予定)
Mol. Cell. Biol. 25・24
Pages: 10953-64
Int. J. Mol. Med. 16・2
Pages: 257-62
Oncogene 24・18
Pages: 3067-72
J. Clin. Invest. 115・4
Pages: 1039-48
Mol. Cell 18・6
Pages: 699-709
J. Biol. Chem. 280・18
Pages: 17694-700
Curr. Eye Res. 30・6
Pages: 437-42
J. Cell Biol. 168・1
Pages: 55-66
J. Biol. Chem. 280・2
Pages: 1095-102
Mol.Cell.Biol. 25
Pages: 10953-10964
Int.J.Mol.Med. 16
Pages: 257-262
Curr.Eye Res. 30
Pages: 437-442
Mol.Cell 18
J.Biol.Chem. 280
Pages: 17694-17700
J.Clin.Invest. 115
Pages: 1039-1048
Oncogene. 24
Pages: 3067-3072
J.Cell Biol. 168
Pages: 1095-1102
Mol.Cell.Biol. 25・24
Int.J.Mol.Med. 16・2
J.Clin.Invest. 115・4
Mol.Cell. 18・6
J.Biol.Chem. 280・18
Curr.Eye Res. 30・6
J.Cell Biol. 168・1
J.Biol.Chem. 280・2
J.Biol.Chem. (発表予定)
Curr.Eye Res. (発表予定)
Dev. Cell 6
Pages: 661-672
Nature Cell Biol. 6
Pages: 1229-1235
J. Biol. Chem. 279
Pages: 46424-46430
Mol. Cell. Biol. 24
Pages: 8386-8394
Genes Dev. 18
Pages: 1824-1837
Invest. Ophthalmol. Vis. Sci. 45
Pages: 2163-2167
J. Virol. 78
Pages: 5338-5346
Anat. Embryol. 208
Pages: 145-150
Graefes Arch. Clin. Exp. Ophthalmol. 242
Pages: 437-441
EMBO J. 23
Pages: 2116-2125
Pages: 9417-9423
Nat.Cell Biol. 6
J.Biol Chem. 279
Mol.Cell.Biol. 24
Invest.Ophthalmol.Vis.Sci. 45
Dev.Cell 6
J.Virol. 78
Graefes Arch.Clin.Exp.Ophthalmol 242
Anat.Embryol 208
J.Biol.Chem. 279
Anat.Embryol. 208
Graefes Arch.Clin.Exp.Ophthalmol. 242