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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 ζ. Expressing TCR-CD3 subunits and tyrosine mutants thereof we tested the specificity of the novel antibodies in Western blot and immuno-purification experiments. In particular, we generated and characterized the monoclonal antibody EM-26 that specifically recognizes phosphorylation of the membrane proximal tyrosine of ζ (phospho-ζY1) and antisera raised against the first and the second phospho-tyrosine of CD3ε (phospho-εY1 and phospho-εY2).
