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Human CAR-3 Reporter Assay Kit

SIZE SKU PRICE
1 x-96 well format assays
3 x-32 assays in-96 well format
1 x-384 well format assays
SIZE SKU
1 x-96 well format assays
3 x-32 assays in-96 well format
1 x-384 well format assays

Product Description and Product Data

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Constitutive Androstane Receptor-3 (CAR-3). INDIGO’s CAR-3 reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the CAR-3. In addition to CAR-3 Reporter Cells, this kit provides two optimized media for use during cell culture and in diluting the user’s test samples, a reference agonist, Luciferase Detection Reagent, and a cell culture-ready assay plate. The principal application of this assay is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert against human CAR-3. This kit provides researchers with clear, reproducible results, exceptional cell viability post-thaw, and consistent results lot to lot. Kits must be stored at -80C. Do not store in liquid nitrogen. Note: reporter cells cannot be refrozen or maintained in extended culture.

Features

  • Clear, Reproducible Results

  • All-Inclusive Assay Systems
  • Exceptional Cell Viability Post-Thaw
  • Consistent Results Lot to Lot

Product Specifications

Target TypeNuclear Hormone Receptor
SpeciesHuman
Receptor FormHybrid
Assay ModeAgonist, Antagonist
Kit Components
  • CAR-3 Reporter Cells
  • Cell Recovery Medium (CRM)
  • Compound Screening Medium (CSM)
  • CITCO (ref. agonist; in DMSO)
  • Detection Substrate
  • Detection Buffer
  • White, sterile, cell-culture ready assay plate
Shelf Life6 months
Orthologs AvailableNo
Shipping RequirementsDry Ice
Storage temperature-80C

Data

Agonist dose-response of the CAR3 Assay. Dose-response analysis of CAR3 Reporter Cells was performed using the reference agonist CITCO (provided). CITCO treatment media were prepared using serial 3-fold dilutions, as described in the Technical Manual. Luminescence was quantified and average relative light units (RLU) and corresponding standard deviation (SD) values were determined for each treatment concentration (n = 3). Values of Fold-Activation and Z’ were calculated. The plot of ‘Fold-Activation vs. Log10[CITCO, nM]’ and EC50 determination were performed via least-squares non-liner regression using GraphPad Prism software.

Target Background

INDIGO’s Human Constitutive Androstane Receptor, isoform 3 (CAR3) Reporter Assay System utilizes proprietary mammalian cells engineered to provide high-level expression of human CAR3 (NR1I3 isoform 3), a ligand-activated transcription factor. The Reporter Cells utilize a modified version of human CAR3 in which the N-terminal DNA binding domain (DBD) has been replaced with the GAL4-DBD. The human CAR3 ligand binding domain (LBD) is unaltered and fully functional. Reporter cells also incorporate a luciferase cDNA functionally linked to the GAL4-upstream activation sequence (UAS). Thus, quantifying expressed luciferase activity provides a sensitive surrogate measure of changes in CAR3 activity resulting from a direct interaction between a treatment compound and the nuclear receptor. Because this assay system expresses a GAL4-DBD + hCAR3 LBD hybrid receptor, the bio-activity of activators that act through indirect mechanisms (such as those that alter the phosphorylation status of the native N-terminal amino acid sequence of CAR3) may be dampened or go undetected.

Human CAR 3 is not constitutively active, rather, it exhibits ligand-dependent activation. The primary application of this reporter assay system is in the screening of test samples to quantify any functional activity, either agonist or antagonist, that they may exert on human CAR3.

Unlike most nuclear receptors, this transcriptional regulator is constitutively active in the absence of ligand but is regulated by both agonists and inverse agonists. Ligand binding results in translocation of this protein to the nucleus, where it activates or represses target gene transcription. These ligands include bilirubin, a variety of foreign compounds, steroid hormones, and prescription drugs.

Citations

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes1-6; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N-acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N-acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh-expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network.
2024-02-27
Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is an organophosphate flame retardant. The primary TDCPP metabolite, bis(1,3-dichloro-2-propyl) phosphate (BDCPP), is detectable in the urine of over 90 % of Americans. Epidemiological studies show sex-specific associations between urinary BDCPP levels and metabolic syndrome, which is an established risk factor for type 2 diabetes, heart disease, and stroke. We used a mouse model to determine whether TDCPP exposure disrupts glucose homeostasis. Six-week old male and female C57BL/6J mice were given ad libitum access to diets containing vehicle (0.1 % DMSO) and TDCPP resulting in the following treatment groups: 0 mg/kg/day, 0.02 mg/kg/day, 1 mg/kg/day, or 100 mg/kg/day. After being on the experimental diet for five weeks without interruption, body composition was analyzed, glucose and insulin tolerance tests were performed, and fasting glucose and insulin levels were quantified. TDCPP at 100 mg/kg/day caused male sex-specific adiposity, fasting hyperglycemia, and insulin resistance. TDCPP-induced modulation of nuclear receptor activation was investigated using an in vitro screen to identify potential mechanisms of metabolic disruption. TDCPP activated farnesoid X receptor (FXR) and pregnane X receptor (PXR), and inhibited the androgen receptor (AR). PXR target genes, but not FXR target genes, were upregulated in livers from mice exposed to 100 mg TDCPP/kg/day. Interestingly, PXR target genes were differentially expressed in livers from both males and females. It remains to be determined whether TDCPP-induced metabolic disruption occurs via modulation of nuclear receptor activity. Taken together, these studies build upon the association of TDCPP exposure and metabolic syndrome in humans by identifying sex-specific effects of TDCPP on glucose homeostasis in mice.
2022-12-07

Also available as a service

Constitutive Androstane Receptor-3 (CAR-3, NR1I3i3)

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