FGFR1c/beta-Klotho Reporter Assay System (mouse)

This is an all-inclusive cell-based luciferase reporter assay kit targeting the Human Hepatocyte Nuclear Factor 4 alpha (NR2A1, HNF4a). INDIGO's HNF4a reporter assay utilizes proprietary mammalian cells that have been engineered to provide constitutive expression of the HNF4a. In addition to HNF4a 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 HNF4a. 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 Type Nuclear Hormone Receptor Species Human Receptor Form Hybrid Assay Mode Agonist, Inverse Agonist Kit Components Human HNF4a Reporter Cells Cell Recovery Medium (CRM) Compound Screening Medium (CSM) CCBA, (ref. agonist, in DMSO) Detection Substrate Detection Buffer White, sterile, cell-culture ready assay plate Shelf Life 6 months Shipping Requirements Dry Ice Storage temperature -80C. Target Background This nuclear receptor assay utilizes proprietary human cells engineered to provide constitutive, high-level expression of the Human Hepatocyte Nuclear Factor 4 alpha (NR2A1), a ligand-dependent transcription factor known as HNF4a. In INDIGO's Reporter Cells the HNF4a receptor is expressed as a hybrid protein in which its native DNA binding domain (DBD) sequence has been exchanged with that of the DBD sequence from the yeast Gal4 gene. Additionally, the cells include the luciferase reporter gene functionally linked to the Gal4 upstream activation sequence (UAS) and a minimal promoter. HNF4a undergoes nuclear translocation, DNA binding, recruitment and assembly of the co-activators and accessory factors required to form a functional transcription complex, culminating in expression of the reporter gene. Thus, quantifying changes in luciferase expression in the treated reporter cells provides a specific and sensitive surrogate measure of the changes in HNF4a activity. Unlike some other assay formats, the readout from INDIGO's reporter cells demands the same orchestration of all intracellular molecular interactions and events that can be expected to occur in vivo. As is true in vivo, the expressed hybrid HNF4a receptor displays a moderate level of constitutively activity. A true ligand interaction may provoke either i.) an agonist response that delivers an additional increase in receptor activity above its already high constitutive level of activity, or ii.) an inverse-agonist response in which a loss of receptor activity is observed. The principal application of this assay is in the screening of test compounds to quantify inverse-agonist or agonist activities that they may exert against the Human HNF4a receptor. These HNF4a Reporter Cells are prepared using INDIGO's proprietary CryoMite(TM) process. This cryo-preservation method yields high cell viability post-thaw, and provides the convenience of immediately dispensing healthy, division-competent reporter cells into assay plates. There is no need for cumbersome intermediate treatment steps such as spin-and-rinse of cells, viability determinations, or cell titer adjustments prior to assay setup. Protein function: Tyrosine-protein kinase that acts as cell-surface receptor for fibroblast growth factors and plays an essential role in the regulation of embryonic development, cell proliferation, differentiation and migration. Required for normal mesoderm patterning and correct axial organization during embryonic development, normal skeletogenesis and normal development of the gonadotropin-releasing hormone (GnRH) neuronal system. Phosphorylates PLCG1, FRS2, GAB1 and SHB. Ligand binding leads to the activation of several signaling cascades. Activation of PLCG1 leads to the production of the cellular signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate. Phosphorylation of FRS2 triggers recruitment of GRB2, GAB1, PIK3R1 and SOS1, and mediates activation of RAS, MAPK1/ERK2, MAPK3/ERK1 and the MAP kinase signaling pathway, as well as of the AKT1 signaling pathway. Promotes phosphorylation of SHC1, STAT1 and PTPN11/SHP2. In the nucleus, enhances RPS6KA1 and CREB1 activity and contributes to the regulation of transcription. FGFR1 signaling is down-regulated by IL17RD/SEF, and by FGFR1 ubiquitination, internalization and degradation. [The UniProt Consortium]