PDK1 Redistribution Assay

• Designed to assay compounds for their ability to modulate membrane translocation PDK1
• 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 3-phosphoinositide-dependent protein kinase 1 (PDK1) functions downstream of phosphoinositide 3-kinase (PI3K) in the PI3K signaling cascade. Upon activation, PDK1 translocates from the cytoplasmic space to the inner surface of the plasma membrane and binds to phospholipids produced by PI3K. Following membrane association, PDK1 initiates complex signaling cascades, including activation of the protein kinase B (PKB)/Akt. Ultimately, PDK1 activity results in cellular activities such as proliferation, differentiation, chemotaxis, survival, trafficking, and glucose homeostasis. The PI3K signaling pathway is activated by growth factors (e.g. insulin and IGF-I).

Figure 1. Membrane translocation of EGFP-PDK1. Cells were treated with 130 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 EGFP-PDK1 detected by the image analysis algorithm.

Figure 2. Concentration response curve in the PDK1 antagonist assay. Wortmannin concentration response curve in the PDK1 antagonist Redistribution assay stimulated by 130 nM IGF-1 (n= 8). The EC₅₀ value of wortmannin is 73 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).