Analysis of novel phospho-ITAM specific antibodies in a S2 reconstitution system for TCR-CD3 signalling.
Immunol Lett
; 130(1-2): 43-50, 2010 May 04.
Article
em En
| MEDLINE
| ID: mdl-20005895
ABSTRACT
The T cell antigen receptor (TCR-CD3) complex contains 12 different cytoplasmic tyrosines, each of which is part of an immunoreceptor tyrosine-based activation motif and thus occurs in similar sequence context. Since phosphorylation of individual tyrosines can be correlated with the quality of the T cell response, monitoring their phosphorylation is important. We thus generated novel antibodies against phospho-tyrosines of the TCR-CD3 complex and tested the specificity in a synthetic biology approach. We utilized the Drosophila S2 reconstitution system testing several kinases and stimulation conditions that lead to optimal phosphorylation of the TCR-CD3 subunit zeta. Expressing TCR-CD3 subunits and tyrosine mutants thereof we tested the specificity of the novel antibodies in Western blot and immunopurification experiments. In particular, we generated and characterized the monoclonal antibody EM-26 that specifically recognizes phosphorylation of the membrane proximal tyrosine of zeta (phospho-zetaY1) and antisera raised against the first and the second phospho-tyrosine of CD3epsilon (phospho-epsilonY1 and phospho-epsilonY2).
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Base de dados:
MEDLINE
Assunto principal:
Linfócitos T
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Transdução de Sinais
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Complexo Receptor-CD3 de Antígeno de Linfócitos T
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Anticorpos Fosfo-Específicos
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article