Journal
NATURE MEDICINE
Volume 10, Issue 12, Pages 1390-1396Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nm1139
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Funding
- NHLBI NIH HHS [N01-HV-28183] Funding Source: Medline
- NIAID NIH HHS [AI50865, AI50854] Funding Source: Medline
- NIAMS NIH HHS [AR49328] Funding Source: Medline
- NIDDK NIH HHS [DK61934] Funding Source: Medline
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We have developed a multiplexed reverse phase protein (RPP) microarray platform for simultaneous monitoring of site-specific phosphorylation of numerous signaling proteins using nanogram amounts of lysates derived from stimulated living cells. We first show the application of RPP microarrays to the study of signaling kinetics and pathway delineation in Jurkat T lymphocytes. RPP microarrays were used to profile the phosphorylation state of 62 signaling components in Jurkat T cells stimulated through their membrane CD3 and CD28 receptors, identifying a previously unrecognized link between CD3 crosslinking and dephosphorylation of Raf-1 at Ser259. Finally, the potential of this technology to analyze rare primary cell populations is shown in a study of differential STAT protein phosphorylation in interleukin (IL)-2-stimulated CD4(+) CD25(+) regulatory T cells. RPP microarrays, prepared using simple procedures and standard microarray equipment, represent a powerful new tool for the study of signal transduction in both health and disease.
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