Protein microarrays for multiplex analysis of signal transduction pathways

Nat Med. 2004 Dec;10(12):1390-6. doi: 10.1038/nm1139. Epub 2004 Nov 21.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • CD3 Complex / metabolism
  • Cross-Linking Reagents / metabolism
  • DNA-Binding Proteins / metabolism
  • Humans
  • Jurkat Cells / metabolism*
  • Jurkat Cells / physiology
  • Kinetics
  • Milk Proteins / metabolism
  • Phosphorylation
  • Protein Array Analysis / methods*
  • Proto-Oncogene Proteins c-raf / metabolism
  • STAT1 Transcription Factor
  • STAT3 Transcription Factor
  • STAT5 Transcription Factor
  • Signal Transduction / physiology*
  • T-Lymphocytes / metabolism
  • Trans-Activators / metabolism


  • CD3 Complex
  • Cross-Linking Reagents
  • DNA-Binding Proteins
  • Milk Proteins
  • STAT1 Transcription Factor
  • STAT1 protein, human
  • STAT3 Transcription Factor
  • STAT3 protein, human
  • STAT5 Transcription Factor
  • Trans-Activators
  • Proto-Oncogene Proteins c-raf