期刊
CELL
卷 162, 期 1, 页码 198-210出版社
CELL PRESS
DOI: 10.1016/j.cell.2015.05.046
关键词
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资金
- USPHS [CA80100, CA82683, CA194584]
- Helmsley Center for Genomic Medicine
- NIH [2 T32 CA009370, R01 NS085296]
- Salk Institute Innovation Grant
- Human Frontiers Science Program
- Genentech Foundation Fellowship for Cancer Research
- NCRR [5P41RR011823-17]
- NIGMS [8P41GM103533-17]
- [P30CA14195]
Histidine phosphorylation (pHis) is well studied in bacteria; however, its role in mammalian signaling remains largely unexplored due to the lack of pHis-specific antibodies and the lability of the phosphoramidate (P-N) bond. Both imidazole nitrogens can be phosphorylated, forming 1-phosphohistidine (1-pHis) or 3-phosphohistidine (3-pHis). We have developed monoclonal antibodies (mAbs) that specifically recognize 1-pHis or 3-pHis; they do not cross-react with phosphotyrosine or the other pHis isomer. Assays based on the isomer-specific autophosphorylation of NME1 and phosphoglycerate mutase were used with immunoblotting and sequencing IgG variable domains to screen, select, and characterize anti-1-pHis and anti-3-pHis mAbs. Their sequence independence was determined by blotting synthetic peptide arrays, and they have been tested for immunofluorescence staining and immunoaffinity purification, leading to putative identification of pHis-containing proteins. These reagents should be broadly useful for identification of pHis substrates and functional study of pHis using a variety of immunological, proteomic, and biological assays.
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