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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.

Graph showing the induction ratios of the promoter reporter constructs of the NFkB Biomarker Set after transfection into HT1080 human fibrosarcoma cells and treatment with TNF-alpha.
Figure 1: Induction ratios for each member of the NFκB Biomarker Set.

HT1080 human fibrosarcoma cells were transfected with the promoter reporter constructs shown, then treated with 20 ng/ml TNF-α or vehicle only. After 8 hours, each sample was assayed using the LightSwitch Luciferase Assay Kit. The graph shows the ratio of treated/untreated signal on a log scale; error bars represent one standard deviation. Please see the heatmap under the Profiling Plate tab below for relative promoter activities under untreated conditions.

*Experimental results and the absolute values of luciferase units may vary depending on cell line, treatment and protocols. All data shown is solely for use as a guide for selecting candidate promoters and panels for your particular experimental design.

 
Name Format Cat No. Price  
LightSwitch™ NFκB Biomarker Set 1 set 32081 $3,500 Buy Now

NFκB Pathway

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.

Applications

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

Diagram of the LightSwitch Luciferase Promoter Reporter Assay.
LightSwitch Promoter Reporter Assays.

To study a promoter's regulation, it is cloned into a LightSwitch Reporter vector upstream of the RenSP luciferase gene and then transfected into mammalian cells. The cells are stimulated, if required, to induce transcription of RenSP luciferase from the cloned promoter. Promoter activity is then quantified using a LightSwitch Luciferase Assay Kit and a luminometer to measure the amount of luminescence produced.

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.

LightSwitch™ NFκB / Inflammation Pathway Profiling Plate

The NFκB Profiling Plate is a subset of promoter reporter constructs chosen from the LightSwitch Promoter Reporter GoClone™ Collection. Promoters that were expected to be up- or down-regulated in the NFκB / Inflammation pathway were chosen based on motif analysis and published information. In-house luciferase testing was then performed to select those promoter reporter constructs that were activated or repressed in response to our induction method, which was to treat the cells with 20 ng/ml TNF-α for 8 hours following transfection.

NFκB Profiling Plate heatmap

The heatmap at right shows the untreated and induced activity for all constructs that are members of the pathway profiling plate. On the right side of the figure, the colors represent log2 transformed ratios of treated/untreated activity according to the color scale shown at the base of the heatmap. Black indicates no change; the intensity of red or blue color indicates levels of induction or repression, respectively. On the left side of the figure, the intensity of yellow color depicts the activity of each promoter in the unstimulated (untreated) condition. All untreated signals are relative to the highest and lowest values within the individual experiment. (“TNF-α” = HT1080 fibrosarcoma cells treated with 20 ng/ml TNF-α for 8 hours).

NFκB Profiling Plate contents

The NFκB Profiling Plate includes 88 promoter reporter constructs (including the 8 top-responding constructs that comprise the NFκB Biomarker Set, as well as 6 housekeeping / random promoters that are unlikely to respond to TNF-α induction). The controls can be used to correct for non-specific changes in overall signal in response to the stimulus. For example, some compounds used for induction are very toxic and result in a significant decrease in overall luciferase signal across the entire panel of constructs in a non-promoter-specific manner. Also included in the plate are 8 transfection controls that are used to determine the conditions that yield maximum transfection efficiency and reproducibility.

View plate map (.xlsx).

Ordering

If you would like to order 5 or fewer members of the NFκB Profiling Plate, you can use the Product IDs found in the file for the plate map above to order directly on Active Motif's SwitchGear Genomics website. If you wish to order the entire plate (or a minimum of 5 or more members), click here to send us an e-mail detailing which constructs you would like, and what amount (5 µg, 10 µg, 1 mg, etc.). We will send you a quote based on your request.

Heatmap data showing the untreated activity and induction response for each construct in the NFkB Pathway Profiling Plate using HT1080 fibrosarcoma cells treated with 20 ng/ml TNF-alpha for 8 hours
Click image to enlarge.

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.