NFkB p65 Redistribution Assay (U2OS)

• Designed to assay compounds for their ability to modulate nuclear translocation of NFκB
• 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-kB (NFkB) is a nuclear transcription factor which regulates the expression of a large number of genes critical for several processes, including apoptosis, viral replication, tumorigenesis, inflammation, and various autoimmune diseases. Activation of NFκB is part of a stress response, and it is activated by growth factors, cytokines, lymphokines, UV light, pharmacological agents, and stress. Five mammalian NF-kB family members are identified (p50, p52, p65, RelB and c-Rel). The transcription factor NF-kB works only when two members form a dimer. The most abundant form consists of a p50 or p52 subunit and a p65 subunit. In its inactive form, NFkB is located in the cytoplasm, bound by members of the IκB family of inhibitor proteins. Stimuli such as interleukin-1β or TNFα cause phosphorylation of IκB, which leads to its ubiquitination and subsequent degradation of the inhibitor protein. This results in nuclear translocation of NFkB and increased NFkB-mediated gene expression. NFκB nuclear translocation can be inhibited by the IκBα specific inhibitor RO 106-9920, which inhibits ubiquitination and degradation of IκBα and subsequent nuclear import of NFkB.

Figure 1. Translocation of NFkB-EGFP in response to TNFα. Cells were treated with 0.25% DMSO (control, left panel) or 10 ng/ml TNFα for 30 min (right panel). The cytoplasm to nucleus translocation is detected by the image analysis algorithm.

Figure 2.  IL-1 and TNFα concentration response curves in the NFkB Redistribution® assay. Concentration response was measured in 9 point half log dilution series of IL-1 or TNFα. Cells were incubated with test samples for 30 min. 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 (10 ng/ml TNFα) and negative control (0.25% DMSO). The EC₅₀ of IL-1 is ~0.07 ng/ml and the EC₅₀ of TNFα is ~0.7 ng/ml.