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Protein microarrays for multiplex analysis of signal transduction pathways


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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Figure 1: RPP microarray performance characteristics and validation.
Figure 2: Application of RPP microarrays in the study of signaling kinetics and pathway delineation.
Figure 3: Protein microarray screen with a panel of 62 phospho-specific antibodies.
Figure 4: CD3 crosslinking induces Raf-1 Ser259 dephosphorylation in both Jurkat and primary human T cells.
Figure 5: Differential STAT protein phosphorylation in IL-2-stimulated CD4+CD25+ TR cells.


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We thank W.H. Robinson and M. Kattah for insightful discussions; and A. Zhang for technical assistance. S.M.C. is supported by the Stanford Medical Scientist Training Program. P.J.U. is supported by grants from the Dana Foundation, Northern California Chapter of the Arthritis Foundation, the Stanford Program in Molecular and Genetic Medicine (PMGM), NIH Grants DK61934, AI50854, AI50865, and AR49328, and NHLBI Proteomics Contract N01-HV-28183.

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Correspondence to Paul J Utz.

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Supplementary information

Supplementary Fig. 1

Representative image of a RPP microarray in an 8-pad subarray format. (PDF 132 kb)

Supplementary Fig. 2

Kinetics of Akt (Ser473), p38 MAPK and SAPK/JNK phosphorylation. (PDF 153 kb)

Supplementary Fig. 3

Differential STAT protein phosphorylation in T regulatory cells in response to IL-2. (PDF 66 kb)

Supplementary Fig. 4

Proof-of-concept experiment demonstrating the use of RPP microarrays for screening compounds with kinase inhibition activity. (PDF 54 kb)

Supplementary Table 1

List of 62 phospho-specific antibodies used in Figure 4 (PDF 8 kb)

Supplementary Note (PDF 6 kb)

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Chan, S., Ermann, J., Su, L. et al. Protein microarrays for multiplex analysis of signal transduction pathways. Nat Med 10, 1390–1396 (2004).

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