EGFP-STAT3 Assay
The EGFP-STAT3 Assay provides a live-cell screening resource to measure the response of the STAT3 signaling pathway. This pathway is important in a number of therapy areas including acute-phase responses, oncology (hematologic, breast, head, neck, and prostate) and obesity. The assay monitors and quantitates the intracellular location of an EGFP-STAT3 fusion protein and its translocation from the cytoplasm to the nucleus when stimulated with an agonist. The assay consists of an extensively validated stably transfected cell line and expression vector. Cells expressing the EGFP-STAT3 protein can be imaged with automated and traditional confocal and wide field epifluorescence microscopes.
- Validated stable cell line: start screening immediately without having to spend months establishing a cell line in-house.
- Expression vector: offers the flexibility to work with transients and alternative host cell lines
- Complete right to use: no additional license negotiations are required prior to using the assay
Move towards a greater understanding>
A measured response>
Results with a range of imaging platforms>
Assay components>
Move towards a greater understanding
Fig 1. Schematic of assay principle.
- Direct quantitation of agonist induced STAT3 movement and location: makes it possible to screen for compounds that have a positive or negative effect on the cascade that leads to the activation of STAT3
- Utilizes Aequorea victoria GFP: the established benchmark fluorescent protein technology
Signal transducers and activators of transcription (STATs) are a family of latent transcription factors that are activated in response to the binding of signaling molecules to cell membrane receptors. Activated STAT proteins then form dimers that translocate to the nucleus and bind to promoter regions in target genes, ultimately modifying the pattern of gene expression.
STAT3 was first isolated from Interleukin (IL) 6 stimulated hepatocytes, but it is has since been shown that STAT3 is activated not only by IL-6, but the entire family of IL-6 type cytokines including IL-11, leukaemia inhibitory factor, ciliary neurotrophic factor, oncostatin M, cardiotrophin-1, and cardotrophin-like cytokine. In addition, other non-cytokine ligands such as epidermal growth factor, hepatocyte growth factor, granulocyte colony-stimulating factor, platelet derived growth factor, granulocyte colonystimulating factor, and leptin have been shown to activate STAT3
and initiate a change in transcription.
STAT3 appears to regulate different genes in different cell types and has been linked to a number of cellular processes including cell proliferation, cell differentiation, and growth arrest. It also has an involvement in diverse processes such as IL-6 dependent T cell proliferation, skin remodeling, deactivation of macrophages and neutrophils, motor neuron survival following axotomy, and mammary gland involution upon forced weaning. More recently STAT3 has been demonstrated to be involved in leptin induced melanocortin production and body energy homostasis, making it an attractive therapeutic target for the control of obesity. It is increasingly apparent that aberrant STAT3 signaling is involved in oncogenesis, making the STAT3 pathway also a target for therapeutic intervention in a number of human cancers.
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A measured response
 | The degree of EGFP-STAT3 translocation is measured using the Nuclear Trafficking Analysis Module. This image analysis routine determines the level of GFP signal (STAT3 expression) throughout the cell and calculates the cytoplasmic-to-nuclear intensity ratio. |
Fig 2. IL-6 dose response curve using the supplied EGFP-STAT3 cell line. Error = ±SD, n = 8 replicates per data point. Image acquisition and data analysis performed using the IN Cell Analyzer 3000.
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Results with a range of imaging platforms
The EGFP-STAT3 assay was designed as a live-cell system using image acquisition and analysis on both the IN Cell Analyzer 3000 and IN Cell Analyzer 1000. However, this assay can also be performed in a fixed-cell format on either of these instruments. Alternatively, the assay may be used in conjunction with other automated subcellular imaging platforms that have appropriate analysis software, or conventional fluorescence microscopes.
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Assay components
- EGFP-STAT3 stably expressing cell line, BHK derived, 2 vials, each containing 1 x 106 cells
- pCORON 1000 EGFP-STAT3 expression vector, 1 vial containing 10-µg DNA
- User manual detailing full assay protocols and validation data
- Rights to use; covering patents relating to GFP, the CMV promoter, and STAT3
* These codes give the user the rights to evaluate the assay for a fixed time period of either 6 or 12 months prior to a full purchase.
Use of the AKT1-EGFP Assay is limited as stated in the terms and conditions of sale.
These vary in accordance with the product code purchased.
EGFP = enhanced green fluorescent protein.
This product can be vsiualised on the IN Cell Analysis System - a total solution for high-content sub-cellular analysis in research and drug discovery.
General Electric Company reserves the right to make changes in specifications and features shown herein, or discontinue the product described at any time without notice or obligation. Contact your GE Representative for the most current information. © 2004 General Electric Company - All rights reserved. Amersham and Amersham Biosciences are trademarks of Amersham plc. Use of this product is limited in accordance with the terms and conditions of sale. Contact us at gfp@amersham.com for details. The Product is developed and sold under license from: BioImage A/S under patents US 6 172 188, EP 851874 and EP 0986753 and other pending and foreign patent applications. Invitrogen IP Holdings Inc (formerly Vertex Pharmaceuticals and Aurora Biosciences Corporation) under US patents 5 625 048, 5 777 079, 5 804 387, 5 968 738, 5 994 077, 6 054 321, 6 066 476, 6 077 707, 6 090 919, 6 124 128, 6 172 188, European patent 1104769 and Japanese patent JP3283523 and other pending and foreign patent applications. Columbia University under US patent Nos. 5 491 084 and 6 146 826. University of Florida Research Foundation under patents US patents 5,968,750, 5,874,304, 5,795,737, 6,020,192 and other pending and foreign patent applications; and Iowa Research Foundation. The CMV promoter is covered under US patents 5 168 062 and 5 385 839 and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation 214 Technology innovation Center Iowa City IA52242 USA; and The Research Institute of The Hospital for Sick Children, Toronto, Ontario, Canada under US patent 6,017,755. Rights to use this product, as configured, are limited to internal use for screening, development and discovery of therapeutic products; NOT FOR DIAGNOSTIC USE OR THERAPEUTIC USE IN HUMANS OR ANIMALS. No other rights are conveyed. Amersham Biosciences UK Limited, a General Electric company, going to market as GE Healthcare.
 | This assay has been developed in collaboration with BioImage.
BioImage is a Danish Biotech company specializing in developing drug
candidates that exert their activity through modulation of protein
translocation. |
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