AKT1-EGFP

AKT1 is a serine/threonine kinase that mediates a number of intra-cellular pathways, including cell survival and insulin signaling. Activation of AKT1 provides a strong survival signal that protects cells from programmed cell death (apoptosis). The AKT1 oncogene has attracted much attention as a drug target because of its central role in regulating cell proliferation and survival. The AKT1-EGFP assay may be used in many application areas, such as oncology, hypertension, and cardiology. The assay consists of an extensively validated stably transfected cell line and expression vector for use with automated and traditional confocal and 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

Fig 1. Schematic of assay principle.

• Direct observation of AKT1 movement and location: permits pathway dissection in correct physiological context, identifies inhibitors of this key cell survival pathway
• Utilizes Aequorea victorea GFP: the established benchmark fluorescent protein technology

One important function of the AKT1 signaling pathway is to promote growth factor-mediated cell survival and protect cells from apoptosis. AKT1 regulates cell survival by directly phosphorylating components of the cell death machinery. Activated AKT1 phosphorylates and deactivates the pro-death Bcl-2 family member BAD; phosphorylated BAD binds 14-3-3, which protects it from dephosphorylation and thus inhibits apoptosis. Caspase 9 proteolytic activity cleaves a variety of cellular substrates that are critical to the generation of the apoptotic phenotype. Inhibition of caspase 9 by AKT-mediated phosphorylation prevents this phenotype evolving. AKT1 is also known to be integrally involved in cellular processes regulating several metabolic enzymes.

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A measured response

The degree of AKT1-EGFP translocation is measured using the Plasma Membrane Spot Analysis Module. This algorithm determines the level of GFP signal (AKT1 expression) throughout the cell and then identifies and quantitates any bright objects (i.e. ruffles) at the edge of the cell.


Fig 2. IGF-1 dose response curve using the supplied AKT1-EGFP cell line. The calculated EC₅₀ was 23 ng/ml. Error = ±SD, n = 8 replicates per data point.

Fig 3. Wortmannin inhibiting curve, in the presence of 1 µg/ml of IGF-1, using the supplied AKT1-EGFP cell line. The calculated EC₅₀ was 15.8 nM. Error = ±SD, n = 4 replicates per data point.

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Results with a range of imaging platforms

AKT1-EGFP assay was optimized for analysis using the IN Cell Analyzer 3000 in conjunction with the Plasma Membrane Spot Analysis Module. It was also developed for image acquisition on the IN Cell Analyzer 1000 and its analysis software (please inquire for availability). Alternatively, the assay may be used in conjunction with other automated subcellular imaging platforms that have appropriate analysis software, or fluorescence microscopes.


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Assay components

• AKT1-EGFP stably expressing cell line, CHO-hIR derived, 2 vials, each containing 1 x 10⁶ cells
• pCORON 1000- AKT1-EGFP 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 and the CMV promoter

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.