NFκB-related Assays (Inflammation)
luciferase reporter constructs validated for inflammation
The LightSwitch™ Validated NFκB Pathway Collection is a comprehensive set of sequence-verified, transfection-ready, promoter reporter constructs that can be used to accurately quantify transcriptional activation and repression in response to induction of the NFκB / Inflammation pathway. These constructs are a subset of the 18,000-member LightSwitch Promoter Reporter GoClone® Collection that were selected based on sequence motif analysis, published information and in-house analysis of each promoter's activity* using our LightSwitch Luciferase Assay.
The NFκB Biomarker Set contains 8 of the highest responding promoters under our chosen test conditions, as well as the promoters of 2 housekeeping genes (Figure 1). The NFκB Profiling Plate contains 88 promoter reporter constructs that showed significant activation or repression in response to induction (See the NFκB Profiling Plate tab below for data and a list of constructs). The IL8, CSF1 and NFKB2 promoter constructs, which are members of both the Biomarker Set and the Profiling Plate, are also available as Pathway Reporter Stable Cell Lines to facilitate high-throughput screening.
Figure 1: Induction ratios for each member of the NFκB Biomarker Set.
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NFκB is a ubiquitous transcription factor that regulates the genes that mediate cell cycle, apoptosis, intercellular communication, immune response and development, memory formation, and tumorigenesis. Various signals such as intracellular stress, cytokines, UV irradiation, free radicals, and infection induce gene expression through the NFκB family. NFκB family members — NFκB1 (p50), NFκB2 (p52), RelA (p65), RelB, and c-Rel share a highly conserved domain responsible for DNA binding to NFκB-elements. Improper regulation of NF-κB has been associated with various cancers, autoimmune diseases, inflammatory disorders, and viral infection. Understanding the NF-kb pathway may aid in identifying preventive and therapeutic measures for diseases, such as chronic inflammation and cancer.
The collection of validated LightSwitch NFκB-related promoter reporters enables you to:
- Understand the mechanisms by which NFκB regulated genes are induced or repressed
- Quantify the functional consequences of transcription factor binding – while many other technologies give a qualitative view of transcription factor level, our promoter reporter assays quantify the effects of transcription factor binding
- Confirm data from ChIP, ChIP-chip or ChIP-Seq experiments
- Measure the effect of sequence variants and mutagenesis on promoter function
- Screen for promoter activation or develop activity profiles across the NFκB signaling pathway for many compounds or conditions in parallel
NFκB in disease: Application highlight
Inflammatory disease: NFκB is a key player in inflammatory disorders such as arthritis, sepsis, and asthma, and inflammatory bowel disease. Many small molecules with anti-inflammatory effects have also been shown to inhibit NFκB. Understanding the activation and repression of genes in the NFκB pathway provides greater value in developing potential therapeutics targeting NFκB in inflammatory disease.
Oncology: As NFκB is a regulator of genes that control cell proliferation, survival, and apoptosis, many tumors have constitutively active NFκB. Blocking NFκB activity has been shown to cease tumor cell proliferation or induce apoptosis. Accordingly, NFκB is the subject of research as a target for anti-cancer therapy.
LightSwitch Luciferase Assay System
The LightSwitch Luciferase Assay System is a complete solution for studying transcriptional & post-transcriptional gene regulation in living mammalian cells. It enables you to directly measure the functional activity of promoters and 3´UTRs through use of an engineered luciferase gene (RenSP) and optimized assay & transfection reagents that ensure superior, reproducible results. In addition to Validated Pathway Collections, the LightSwitch System includes collections of over 30,000 cloned human promoter reporter constructs and human 3´UTR reporter constructs, miRNA mimics and inhibitors, stable cell lines, as well as collections of synthetic long-range response element constructs and synthetic 3´UTRs target validation constructs that can provide more sensitivity than endogenous sequences. Comprehensive Custom Services are also available
LightSwitch Promoter Reporter Assays.
Advantages of the LightSwitch Luciferase Assay System
- Quantitative – Novel RenSP luciferase technology allows you to measure promoter activity with industry-leading sensitivity and dynamic range.
- Simple, fast, complete solution – With pre-cloned LightSwitch Reporter vectors and optimized transfection & assay reagents, you can study regulation of your gene today. No cloning, DNA preparation or optimization is needed, and most studies do not require any internal transfection controls.
- Comprehensive and verified – The genome-wide LightSwitch Reporter Collections are sequence-verified, transfection-ready promoter and 3´UTR reporter vectors.
- Functionally insightful – Learn about the actual effects of transcription factor binding to verify computational predictions and supplement microarray or sequencing data.
- Cost-effective – Efficiently screen for activation and/or repression using a multitude of conditions.
The NFκB Biomarker Set contains 5 µg aliquots each of 8 promoter reporter constructs and 2 housekeeping promoter constructs (ACTB & RPL10) for use as controls. Click on the links below for clone information.
The NFκB Profiling Plate includes 88 promoter reporter constructs and 8 transfection control constructs (4 constructs from housekeeping genes with promoter activities of varying strengths (P1-P4), and 4 constructs with random 1 kb fragments (R1-R4)). To download a plate map with links to clone information, please click here.
** The IL8, CSF1 and NFKB2 promoter constructs, which are members of both the Biomarker Set and the Profiling Plate, are also available as Pathway Reporter Stable Cell Lines to facilitate high-throughput screening.