Akt3 Redistribution Assay

• Designed to assay compounds for their ability to modulate membrane translocation of Akt3
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

The Akt/protein kinase B (PKB) family of serine/threonine-specific protein kinases comprises three highly homologous members in mammalian cells (Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ). The Akt family members are activated by diverse stimuli such as hormones and growth factors (e.g. insulin and IGF-I). The Akt protein kinases function within the phosphoinositide 3-kinase (PI3K) signaling pathway. PI3K generates phosphatidylinositol-3,4,5-trisphosphate (PIP3), a lipid second messenger essential for the translocation of Akt/PKB to the plasma membrane. Following translocation to the membrane, Akt is phosphorylated and activated by the phosphoinositide-dependent kinase-1 (PDK-1). Activated Akt phosphorylates and regulates the function of many cellular proteins essential for metabolism, apoptosis, and proliferation.

Figure 1. Membrane translocation of Akt3-EGFP. Cells were treated with 100 nM IGF-1 without (DMSO control, left panel) and with (right panel) addition of 300 nM wortmannin. Arrows indicate IGF-1 induced membrane translocation of Akt3-EGFP detected by the image analysis algorithm.

Figure 2. Concentration response curve in the Akt3 antagonist assay. Wortmannin concentration response curve in the Akt3 antagonist Redistribution assay stimulated by 100 nM IGF-1 (n=16). The EC₅₀ of wortmannin is 35 nM. Concentration response was measured in 9 point half log dilution series. Cells were pre-incubated with 100 nM IGF-1 for 60 min. and treated with wortmannin for 4 min. Cells were then fixed and membrane translocation was measured using the Cellomics ArrayScan V^TI Reader and the CytoCellMemTrans.V2 BioApplication. % activity was calculated relative to the positive (300 nM wortmannin) and negative control (0.25% DMSO).