NFkB p65 Redistribution Assay (CHO)

• Designed to assay compounds for their ability to modulate activation of NFkB
• 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 NFkB is part of a stress response, and it is activated by growth factors, cytokines, lymphokines, UV light, pharmacological agents, and stress. Five mammalian NFkB 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 IkB family of inhibitor proteins. Stimuli such as interleukin-1β or TNFα cause phosphorylation of IkB, which leads to its ubiquitination and subsequent degradation of the inhibitor protein. This results in nuclear translocation of NFkB p65 and increased NFkB-mediated gene expression. NFkB p65 nuclear translocation can be inhibited by the IkBα specific inhibitor RO 106-9920, which inhibits ubiquitination and degradation of IkBα and subsequent nuclear import of NFkB p65.

Figure 1. Translocation of NFkB-3FP stimulated with IL-1 in response to RO106-9920. Cells were treated with 5 ng/mL IL-1 in the absence (DMSO control, left panel) or presence (right panel) of 50 μM RO106-9920. Arrows indicate IL-1-mediated nuclear translocation detected by the image analysis algorithm.

Figure 2. RO106-9920 concentration response curve in the NFkB Redistribution assay. Concentration response was measured in 9 point half log dilution series of RO106-9920. Cells were then fixed and the nucleus to cytoplasm translocation was measured using the Cellomics ArrayScan® V^TI Reader and RedistributionV3 BioApplication. % activity was calculated relative to the positive (50 μM RO106-9920) and negative control (0.25% DMSO). The EC₅₀ value of RO106-9920 is approximately 5 μM in the assay.