EGFP-SMAD2 Assay
The EGFP-SMAD2 Assay provides a live-cell screening resource to measure the response of the Transforming Growth Factor (TGF)-b signaling pathway; imbalance or disruption of which has implications in many cellular processes such as proliferation, apoptosis, and cell growth. The assay monitors and quantitates the intracellular location of an EGFP-SMAD2 fusion protein and its translocation from the cytoplasm to the nucleus following activation by TGF-b1. The assay consists of an extensively validated stably transfected cell line and expression vector. Cells expressing the EGFP-SMAD2 reporter 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
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Unstimulated cell
EGFP-SMAD2 is localized in the cytoplasm | Unstimulated cell
EGFP-SMAD2 translocates to the nucleus |
Fig 1. Schematic of assay principle.
- Direct quantitation of TGF-b induced SMAD2 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 SMAD2
- Utilizes Aequorea victorea GFP: the established benchmark fluorescent protein technology
The TGF-b superfamily comprises peptide growth factors known to exert a wide spectrum of biological responses on a large number of cell types in both vertebrates and invertebrates. Many of these effects have important roles in embryonic development and adult homeostasis, being involved in cell growth, proliferation, apoptosis, extracellular matrix secretion and adhesion, terminal differentiation, and specification of developmental fate. Transcriptional control by the TGF-b superfamily is an area of intense research, since imbalance or disruption of critical SMAD-mediated pathways has been implicated in many disease states such as cancer (breast, prostate, ovarian, and gastric), tumor progression, drug and radiation resistance in cancer therapy, invasion, angiogenesis, and diabetes.
SMAD proteins themselves comprise a family of transcription factors ubiquitously expressed throughout development in all adult tissues in vertebrates, insects, and nematodes. Receptor activation results in phosphorylation and hetero-oligomerization of downstream SMADs, which subsequently translocate to the nucleus. SMADs 2 and 3 mediate TGF-b/Activin signaling and are the only TGF-b substrates with a demonstrated ability to propagate signals.
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A measured response
 | The degree of EGFP-SMAD2 translocation is measured using the Nuclear Trafficking Analysis Module; this analysis module quantitates the degree of pathway response by quantitating the nucleus-to-cytoplasm intensity ratio of the EGFP-SMAD2 fusion protein in cells challenged with test compounds. The assay has been validated in an agonist format using TGF-b1, generating a typical EC50 value of 3 pM. |
Fig 2. TGF-ß1 dose response curve. Data acquired and analyzed using the IN Cell Analyzer 3000. Error = ± SD, n = 8 replicates per data poin.
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Results with a range of imaging platforms
The EGFP-SMAD2 assay was designed as a live-cell system that can be imaged and analyzed on either the IN Cell Analyzer 3000 or IN Cell Analyzer 1000. Alternatively, the assay may be used in conjunction with other automated subcellular imaging instruments that have appropriate analysis software, or conventional epifluorescence microscopes.
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Assay components
- EGFP-SMAD2 stably expressing cell line, CHO-hIR derived, 2 vials, each containing 1 x 106 cells
- pCORON 1000- EGFP-SMAD2 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 SMAD2
* 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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