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Profiling Kinase Pathway Activation and Inhibitor Activity Using a Novel Antibody Array
Author(s) -
Radding Jeffrey Alan,
RauhAdelmann Christine,
Hall Amy B.,
Murphy Cheryl,
Yen Lucy G.,
Graham James R.,
Nadler Timothy K.,
Gordon Neal F.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1054.2
Subject(s) - phosphorylation , signal transduction , antibody microarray , tyrosine kinase , kinase , protease , cell culture , peptide , microbiology and biotechnology , cell , phosphoproteomics , biology , chemistry , protein kinase a , protein phosphorylation , biochemistry , antibody , enzyme , immunology , genetics
Broad screening of protein phosphorylation has wide‐ranging utility for understanding the impact of disease or drug treatment on cell signaling pathways. To address this need, we have developed an antibody array for measuring tyrosine signaling across 45 phosphotyrosine sites across multiple signaling pathways (Ti‐Tyr ™ Profiling Chip). The approach utilizes a novel strategy of measuring peptide fragments liberated by protease digestion of the sample prior to analysis, and allows for both multi‐site profiling and absolute quantification using synthetic peptide standards. The utility of the Ti‐Tyr™ chip is demonstrated by measuring specific modulation of pathway phosphorylation in stimulated cells in the presence and absence of distinct kinase inhibitors. Phosphorylation changes detected in chip targets correlate with known biological effects using comparable conditions. The use of peptide standards on the chip allows for an absolute quantitative assessment of the same stimulation conditions or inhibitor treatments across different cell lines, for example EGF stimulation in A431 and PC‐3 cancer lines. The Ti‐Tyr ™ Tyrosine Chip should improve the ability to understand the effects of cell stimuli, the underlying basis of disease and the impact of drug treatments across multiple cell pathways and cell types.