| Immunofluorescent quantification of tyrosine phosphorylation of cellular proteins in whole cells by flow cytometry. | |
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MedLine Citation:
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PMID: 7517816 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Tyrosine phosphorylation of proteins, a major event in the transduction of mitogenic signals, was analysed by flow cytometry with a fluorescent antiphosphotyrosine monoclonal antibody, on formaldehyde-fixed, permeabilized cells. We have used this method (PY-Facs) to study activation of normal human T lymphocytes and cells of a leukemic T-cell line: Jurkat. In contrast to normal T cells, Jurkat cells as well as three other leukemic cell lines display a higher constitutive level of tyrosine phosphorylation. This level of tyrosine phosphorylation results from an equilibrium that can be up-regulated by the tyrosine phosphatase inhibitor, vanadate peroxide, and down-regulated by the tyrosine kinase inhibitors, genistein and staurosporine. We have also observed an increased tyrosine phosphorylation of proteins after mitogenic stimulation of Jurkat cells via T-cell receptor triggering. In addition, the entry of normal purified T cells from G0 phase into the cell cycle after co-stimulation with a phorbol ester and an anti-receptor antibody is correlated with a pronounced increase in tyrosine phosphorylation. We thus confirmed that this biochemical event was tightly associated with the activation status of the cells. The rapidity and sensitivity of the method we describe here make it particularly convenient for routine use and processing of a large number of samples, e.g., during analysis of human tumors. Moreover, because it retains sufficiently the integrity of treated cells and does not alter expression of membrane antigens, this method is suitable for multiparametric analysis, particularly for simultaneous studies associating the measure of tyrosine phosphorylation levels with possible modifications of membrane or intracellular structures as well as with cell cycle status.(ABSTRACT TRUNCATED AT 250 WORDS) |
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Authors:
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D F Far; J F Peyron; V Imbert; B Rossi |
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Publication Detail:
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Type: Comparative Study; Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Cytometry Volume: 15 ISSN: 0196-4763 ISO Abbreviation: Cytometry Publication Date: 1994 Apr |
Date Detail:
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Created Date: 1994-08-08 Completed Date: 1994-08-08 Revised Date: 2009-11-19 |
Medline Journal Info:
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Nlm Unique ID: 8102328 Medline TA: Cytometry Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 327-34 Citation Subset: IM |
Affiliation:
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INSERM U364, Faculté de Médecine, Nice, France. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Antibodies, Monoclonal Blotting, Western Enzyme Activation Flow Cytometry* Fluorescent Antibody Technique* Humans Leukemia, T-Cell / pathology* Lymphocyte Activation Muromonab-CD3 / pharmacology Neoplasm Proteins / analysis*, metabolism Phosphoproteins / analysis* Phosphorylation Phosphotyrosine Protein Processing, Post-Translational* Protein-Tyrosine Kinases / metabolism Proteins / metabolism Sensitivity and Specificity Signal Transduction T-Lymphocytes / chemistry* Tumor Cells, Cultured Tyrosine / analogs & derivatives*, analysis |
| Chemical | |
Reg. No./Substance:
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0/Antibodies, Monoclonal; 0/Muromonab-CD3; 0/Neoplasm Proteins; 0/Phosphoproteins; 0/Proteins; 21820-51-9/Phosphotyrosine; 55520-40-6/Tyrosine; EC 2.7.10.1/Protein-Tyrosine Kinases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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