NFATc1 Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of NFATc1
• 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

Nuclear factor of activated T-cells c1 (NFATc1) is a transcription factor involved in T-cell signaling and tissue development, and its activity is controlled by the Ca²⁺ Calmodulin-dependent phosphatase, calcineurin. Inactive NFATc1 transcription factor resides in the cytosol. In response to sustained elevated calcium levels, NFATc1 is dephosphorylated by calcineurin, which induces its rapid translocation to the nucleus. In the nucleus it forms transcription complexes with other transcription factors such as AP-1, GATA4, GATA2, and MEF2. NFATc1 dephosphorylation and nuclear translocation can be inhibited pharmacologically by the microbial products FK506 and cyclosporin A (CsA). These reagents bind to the intracellular proteins FKBPK and cyclophilin respectively, and they subsequently bind to calcineurin and block phosphatase activity.

Figure 1. Images of cells stably transfected with EGFP-NFATc1. Cells have been treated with 1 µM ionomycin in the absence (DMSO control, left panel) or presence (right panel) of 300 nM FK506 antagonist. Antagonist treatment leads to nucleus to cytoplasm translocation of EGFP-NFATc1.

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Figure 2. Concentration response curves in the NFATc1 Redistribution assay. Concentration response was measured in 9 point dilution series of cyclosporine A and FK506 (n=4). Cells were incubated with test compound for 65 min before treatment with 1 µM ionomycin for 5 min. Cells were then fixed and the nucleus tocytoplasm translocation was measured using image analysis. % activity was calculated relative to the positive (300 nM FK506) and negative control (0.25% DMSO). The EC₅₀ values are: FK506 EC₅₀=2.4 nM, Cyclosporine A EC₅₀=18 nM