STAT1 Redistribution Assay

• Designed to assay compounds for their ability to modulate nuclear translocation of STAT1
• 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

STAT1 (Signal transducer and activator of transcription 1) plays a crucial role in signaling by interferons (IFNs) thereby regulating antiviral responses, cell proliferation, apoptosis, immune surveillance, and tumor suppression. STAT1 transmits signals from the membrane to the nucleus, and binding of ligands such as the cytokine γ-interferon (IFNγ) to its receptor activates the Janus kinase (JAK)/STAT signalling pathway. Inactive STAT1 is cytoplasmic, but upon activation it is rapidly recruited to activated receptors, where its association with JAK catalyzes ligand-induced phosphorylation of STAT1. This leads to an SH2-mediated dimerization of STAT1, followed by translocation to the nucleus and activation of cytokine-responsive genes. STAT1 nuclear translocation can be inhibited by JAK inhibitor 1, thereby blocking STAT1 phosphorylation and subsequent nuclear import.

Figure 1. Translocation of STAT1-EGFP. Cells were treated with 200 ng/mL γ-interferon in the absence (left panel) or presence (right panel) of 1 μM JAK inhibitor 1. Arrows indicate γ-interferon-mediated nuclear translocation detected by the image analysis algorithm.

Figure 2. JAK inhibtor 1 concentration response in the STAT1 Redistribution assay. Concentration response was measured in 9 point half log dilution series (n = 12). Cells were then fixed and the nucleus to cytoplasm translocation was measured using the Cellomics ArrayScan® V^TI Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (1 μM Jak Inhibitor 1) and negative control (0.25% DMSO). The EC₅₀ of Jak Inhibitor 1 is ~30 nM.