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LanthaScreen™ STAT1 U2OS Cell Line

The JAK/STAT1 signaling pathway is known to be activated by type I/II interferons such as interferon-gamma and interferon-alpha. In this pathway, binding of these cytokines to their respective cell-surface receptors results in the activation of JAKs, which in turn phosphoactivate STAT1 proteins at a specific tyrosine residue (Y701). LanthaScreen® STAT1 U2OS is a human cell line that constitutively expresses a GFPSTAT1 fusion protein. The JAK/STAT1 signaling pathway is known to be functionally intact in this cell line; therefore, the GFP-STAT1 fusion protein serves as a substrate for the IFN-gamma-inducible phosphorylation by JAKs. Using this cell line, a homogeneous immunoassay has been developed in which the phosphorylation state of GFP-STAT1 is detected in cell lysates using a terbium-labeled anti-pY701-STAT1 antibody in a time-resolved FRET (TR-FRET) readout. The GFP-STAT1 DNA expression construct was transfected into U2OS cells using Lipofectamine™ LTX Reagent, followed by selection with blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as a sorting marker. Using the lytic TR-FRET immunoassay, this cell line is validated for EC50 and Zfi-factors under optimized conditions using IFN-gamma as a ligand for JAK-mediated GFP-STAT1 phosphorylation. This assay has also been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance, and assay development time. Additional information using alternate stimuli and alternate assay protocols is available.

GeneBLAzer™ P2RY6-NFAT-bla CHO-K1 Cells

GeneBLAzer® P2RY6-NFAT-bla CHO-K1 cells contain the human Purinergic receptor P2, G protein-coupled, 6 (P2RY6) stably integrated into the CellSensor® NFAT-bla CHO-K1 cell line. CellSensor® NFAT-bla CHO-K1 cells (Cat. no. K1534) contain a beta-lactamase reporter gene under control of the Nuclear Factor of Activated T-cells (NFAT) response element.

The GeneBLAzer® P2RY6-NFAT-bla CHO-K1 cells are functionally validated for Z' and EC50 concentrations of UDP. In addition, GeneBLAzer® P2RY6-NFAT-bla CHO-K1 cells have been tested for assay performance under variable conditions. These data are found in the Validation & Assay Performance Summary.

P2RY6 has been found in a variety of rat tissue such as; placenta, spleen, thymus, lung, stomach, intestine, mesentery, heart, and aorta. P2RY6 is activated by UDP, which functions as a full agonist. P2RY6 is activated either weakly or not at all by additional extracellular nucleotides such as UTP, ATP, and ADP. P2RY6 has been implicated to be involved in ion transport, vasodilatation, and contraction of smooth muscle.

For Academic pricing please login or contact us at discoverysciences@invitrogen.com.

GeneBLAzer™ ERR Alpha DA Assay Kit

GeneBLAzer®ERR alpha DA(Division Arrested) cells and ERR alpha-UAS-bla HEK 293T cells contain the ligand-binding domain (LBD) of the human Estrogen related receptor alpha (ERR alpha) fused to the DNA-binding domain of GAL4 stably integrated in the GeneBLAzer®UAS-bla HEK 293T cell line. GeneBLAzer®UAS-bla HEK 293T cells stably express a beta-lactamase reporter gene under the transcriptional control of an upstream activator sequence (UAS). When an agonist binds to the LBD of the GAL4 (DBD)-ERR alpha (LBD) fusion protein, the protein binds to the UAS, resulting in expression of beta-lactamase. Division Arrested (DA) cells are available in two configurations- an Assay Kit (which includes cells and sufficient substrate to analyze 1 x 384-well plate), and a tube of cells sufficient to analyze 10 x 384-well plates. DA cells are irreversibly division arrested using a low-dose treatment of Mitomycin-C, and have no apparent toxicity or change in cellular signal transduction. Both ERR alpha DA cells and ERR alpha-UAS-bla HEK 293T cells are functionally validated for Z' and EC50 concentrations of XCT790, an inverse agonist. In addition, ERR alpha-UAS-bla HEK 293T cells have been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time (data available upon request). Additional testing data using alternate stimuli are also available.

GeneBLAzer™ LXR beta DA Assay Kit

GeneBLAzer®LXR beta DA (Division Arrested) cells and LXR beta-UAS-bla HEK 293T cells contain the ligand-binding domain (LBD) of the human Liver-X receptor-beta (LXR beta) fused to the DNA-binding domain of GAL4 stably integrated in the GeneBLAzer®UAS-bla HEK 293T cell line. GeneBLAzer®UAS-bla HEK 293T cells stably express a beta-lactamase reporter gene under the transcriptional control of an upstream activator sequence (UAS). When an agonist binds to the LBD of the GAL4 (DBD)-LXR beta (LBD) fusion protein, the protein binds to the UAS, resulting in expression of beta-lactamase. Division Arrested (DA) cells are available in two configurations- an Assay Kit (which includes cells and sufficient substrate to analyze 1 x 384-well plate), and a tube of cells sufficient to analyze 10 x 384-well plates. DA cells are irreversibly division arrested using a low-dose treatment of Mitomycin-C, and have no apparent toxicity or change in cellular signal transduction. Both LXR beta DA cells and LXR beta-UAS-bla HEK 293T 293 cells are functionally validated for Z' and EC50 concentrations of TO901317. In addition, LXR beta-UAS-bla HEK 293T 293 cells have been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time (data available upon request). Additional testing data using alternate stimuli are also available.

LanthaScreen™ c-Jun (1-79) HeLa Cell Line

The LanthaScreen®c-Jun (1-79) HeLa cell line contains a fusion protein consisting of GFP and a fragment encoding for AA 1-79 of c-Jun under the control of a CMV promoter stably transfected into HeLa cell lines. HeLa is a cervical cancer cell line which shows inducible activation of JNK by a number of different ligands, such as TNF-a and EGF, which leads to the transient phosphorylation of GFP-c-Jun (1-79). This LanthaScreen®cell line therefore allows the analysis of JNK activity using GFP-c-Jun phosphorylation as readout. The GFP-c-Jun (1-79) construct was transfected into HeLa cells using Lipofectamine™2000, followed by selection with Blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as sorting marker. This cell line is validated for EC50 and Z' under optimized conditions using TNF-a as ligand for JNK mediated GFP-c-Jun (1-79) phosphorylation. This cell lines has also been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance and assay development time. Additional information using alternate stimuli is also available. Academic and non-profit customers, please inquire for special pricing.

CellSensor™ TrkB-NFAT-bla CHO-K1 Cell Line

Neurotrophins (NGF, BDNF, NT-3, and NT-4) and their transmembrane receptors (TrkA, TrkB, TrkC, and P75NTR) play important roles in the regulation of neuronal and non-neuronal cell proliferation, differentiation, survival, and death. Neurotrophin signaling also mediates higher-order neuronal activities, such as learning, memory, and behavior. Alterations in neurotrophin levels and their receptors have been implicated in neurodegenerative disorders, such as Alzheimer's disease and Huntington's disease, as well as psychiatric disorders. Neurotrophins propagate their signal through activating multiple signaling pathways (e.g., MAPK, PI3K, PKC).

BDNF (brain-derived neurotrophic factor) and its receptor TrkB (also known as NTRK2) play a fundamental role in regulating neural development, survival, and synaptic activity and plasticity. TrkB is also a potential anticancer target, since altered signaling through TrkB promotes tumor formation, survival, and metastasis of various cancers (including neuroblastomas, multiple myelomas, and pancreatic ductal adenocarcinomas). Moreover, growing evidence indicates that BDNF and its receptor influences food intake and body weight control.

CellSensor® TrkB-NFAT-bla CHO-K1 cells contain a beta-lactamase reporter gene under control of the NFAT Response Element that has been stably integrated into CHO-K1 cells along with TrkB. CellSensor® TrkB-NFAT-bla CHO-K1 cells express beta-lactamase upon stimulation with brain-derived neurotrophic factor (BDNF). This cell line is a clonal population isolated by flow cytometry and has been tested for robust performance by assessing a variety of assay parameters.

GeneBLAzer™ PPARγ-UAS-bla HEK 293H Cells

PPAR gamma-UAS-bla HEK 293H cells contain the ligand-binding domain (LBD) of the human Peroxisome Proliferator-Activated Receptor-gamma (PPAR gamma) fused to the DNA-binding domain of GAL4 stably integrated in the GeneBLAzer®UAS-bla HEK 293H cell line. GeneBLAzer®UAS-bla HEK 293H cells stably express a beta-lactamase reporter gene under the transcriptional control of an upstream activator sequence (UAS). When an agonist binds to the LBD of the GAL4 (DBD)-PPAR gamma (LBD) fusion protein, the protein binds to the UAS, resulting in expression of beta-lactamase. Division Arrested (DA) cells are available in two configurations- an Assay Kit (which includes cells and sufficient substrate to analyze 1 x 384-well plate), and a tube of cells sufficient to analyze 10 x 384-well plates. PPAR gamma-UAS-bla HEK 293H cells are functionally validated for Z' and EC50 concentrations of Rosiglitazone. In addition, PPAR gamma-UAS-bla HEK 293H cells have been tested for assay performance under variable conditions, including DMSO concentration, cell number, and stimulation time.

LanthaScreen™ Akt HEK 293E Cell Line

LanthaScreen® GFP Cellular Assays allow for the analysis of post-translational modifications for a number of target proteins in an live-cell context. The PI3K⁄AKT pathway mediates signals for cell growth, cell survival, transcription, translation, and glucose uptake. Because of the complexity of this signaling cascade, especially as applied to the regulation of the mammalian target of rapamycin (mTOR), cell-based methods are critical for proper identification of small-molecule mediators of this pathway. The significance of mTOR as a kinase has been underscored recently by the identification of two distinct multimeric complexes inside the cell: mTORC1 (includes raptor and is rapamycin sensitive) and mTORC2 (includes rictor and is insensitive to acute rapamycin exposure). mTORC2 has been shown to phosphorylate and AKT at residue Ser473 for complete activation of this prosurvival kinase. LanthaScreenTM AKT HEK293E is a human cell line which constitutively expresses GFP-AKT fusion proteins. This kinase target was introduced using lipid transfection and these cells are a clonal population isolated by FACS, using GFP fluorescence as a sorting marker and Blasticidin to maintain cells under selection. Using this cell line, a homogenous immunoassay was developed with a time-resolved FRET (TR-FRET) readout in which the insulin-induced phosphorylation of Ser473 on GFP-AKT is detected in cell lysates using a terbium-labeled phosphor-specific antibody. This cell line has been validated with different stimuli⁄inhibitors and shows correct EC50 ⁄ IC50 values. Moreover, this assay has been optimized for performance under variable experimental conditions (including cell plating density, agonist stimulation time, DMSO tolerance and assay development time) and displays excellent statistical data (Z' > 0.6) and good signal-to-background.

Tango™ TBXA2R-bla U2OS Cells

The Tango™ TBXA2R-bla U2OS cells contain the human Thromboxane A2 Receptor (TBXA2R) linked to a TEV protease site and a Gal4-VP16 transcription factor stably integrated into the Tango™ GPCR-bla U2OS parental cell line. This parental cell line stably expresses a beta-arrestin⁄TEV protease fusion protein and the beta-lactamase (bla) reporter gene under the control of a UAS response element.

The Tango™ TBXA2R-bla U2OS cells cells have been functionally validated for Z' factor and EC50 concentrations of established ligands. In addition, Tango™ TBXA2R-bla U2OS cells have been tested for assay performance under variable conditions. These data are found in the Validation & Assay Performance Summary for each product.

CellSensor™ c-Fos-bla ME-180 Cell Line

The CellSensor® c-fos-bla ME-180 cell line contains a beta-lactamase reporter gene under control of the c-fos response element stably integrated into ME-180 cells. To construct this cell line, the c-fos-bla construct was transduced into ME-180 cells by lentivirus. Flow cytometry was used to isolate cells responsive to epidermal growth factor (EGF). This cell line is validated for DMSO tolerance, cell number, stimulation time, substrate loading time, as well as Z' and EC50 concentrations for EGF. The CellSensor® c-fos-bla ME-180 cell line is responsive to EGF, HGF, IL-6, OSM, TNFα, IL-1-alpha; and PMA/Thaps, and can be used to probe the JNK/P38/MAPK and JAK/STAT signaling pathways. This cell line can be adapted for high-throughput screening of agonist or antagonist compound libraries. Candidate drugs can also be tested for dose response against this cell line. Academic and non-profit customers, please inquire for special pricing.

GeneBLAzer™ M1 CHO-K1 DA Assay Kit

This cell line is available as dividing cells by requesting a quote for K1230 from dividingcells@invitrogen.com

GeneBLAzer® M1 CHO-K1 DA(Division Arrested) cells and M1-NFAT-bla CHO-K1 cells contain the human Acetylcholine (muscarinic) subtype 1 receptor (M1) stably integrated into the CellSensor® NFAT-bla CHO-K1 cell line. CellSensor® NFAT-bla CHO-K1 cells (Cat. no.K1078) contain a beta-lactamase reporter gene under control of the Nuclear Factor of Activated T-cells (NFAT) response element. Division Arrested (DA) cells are available in two configurations: an Assay Kit (which includes cells and sufficient substrate to analyze 1 x 384-well plate), and a tube of cells sufficient to analyze 10 x 384-well plates.

DA cells are irreversibly division arrested using a low-dose treatment of Mitomycin-C, and have no apparent toxicity or change in cellular signal transduction. Both M1 CHO-K1 DA cells and M1-NFAT-bla CHO-K1 cells are functionally validated for Z’-factor and EC50 concentrations of carbachol, (Figure 1). In addition, M1-NFAT-bla CHO-K1 cells have been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time (data available upon request). Additional testing data using alternate stimuli are also available.

The Musacarinic 1(M1) Receptor is localized principally in the cortical and hippocampal regions of the brain and has been implicated in various neurological conditions including depression, anxiety, and sleep disorders.


Storage:

GeneBLAzer® M1 CHO-K1 DA Cells and M3-NFAT-bla CHO-K1 Cells are shipped on dry ice. Store in liquid nitrogen immediately upon receipt or thaw for immediate use.


Contents:
GeneBLAzer® M1 CHO-K1 DA Assay Kit
Each system contains sufficient division arrested (DA) cells & substrate to assay 1 x 384-well plate. (Other materials are required separately; please refer to the protocol).
Includes:
•M1 CHO-K1 DA cells
•LiveBLAzer™-FRET B/G Loading Kit, 70µg
•Solution D, 1ml
•2 protocols
•Certificate of Analysis
GeneBLAzer® M1 CHO-K1 DA cells
Each system contains sufficient division arrested (DA) cells to assay 10 x 384-well plates (LiveBLAzer™-FRET B/G Loading Kit, Solution D and other materials are required separately; please refer to the protocol)..
Includes:
•M1 CHO-K1 DA cells
•1 protocol
•Certificate of Analysis

GeneBLAzer® M1-NFAT-bla CHO-K1 cells
(LiveBLAzer™-FRET B/G Loading Kit, Solution D and other materials are required separately; please refer to the protocol).
Includes:
•M1-NFAT-bla CHO-K1 cells
•1 protocol
•Certificate of Analysis

CellSensor™ AP-1-bla ME-180 Cell Line

The CellSensor® AP-1-bla ME-180 Cell Line contains a beta-lactamase reporter gene under control of the AP-1 response element stably integrated into ME-180 cells. The cell line was created through FACS sorting of cells responsive to stimulation of the AP-1 pathway with Epidermal Growth Factor (EGF). This cell line was validated for DMSO tolerance, stimulant incubation time, substrate loading conditions, Z' and EC50 concentration of Epidermal Growth Factor. The AP-1-bla ME-180 Cell Line responds to agonist treatment as expected from the literature and can be adapted for high-throughput screening for agonists or antagonists of the AP-1 pathway with compound libraries. Candidate drugs can also be tested for dose response against this cell line. Academic and non-profit customers, please inquire for special pricing.

GeneBLAzer™ RAR alpha DA Assay Kit

The GeneBLAzer®RAR alpha DA (Division Arrested) and RAR alpha-UAS-bla HEK 293T cells contain the ligand-binding domain (LBD) of the human retinoic acid receptor alpha fused to the DNA-binding domain of GAL4 stably integrated in the GeneBLAzer®UAS-bla HEK293T cell line. GeneBLAzer®UAS-bla HEK 293T cells (catalog#K1104) stably express a beta-lactamase reporter gene under the transcriptional control of a 7x Upstream Activator Sequence (UAS). Transcription from the 7xUAS is activated by the binding of the GAL4 transcription factor DNA-binding-domain (DBD). The GAL4-DBD is expressed as a fusion protein with the ligand binding domain (LBD) of RAR alpha. When an agonist binds to the LBD of the GAL4(DBD)-RAR alpha(LBD) fusion protein it translocates to the nucleus where it binds to the 7x UAS inducing transcription of beta-lactamase. Division Arrested (DA) cells are available in an Assay Kit (which includes cells and sufficient substrate to analyze 1 x 384-well plate). DA cells are irreversibly division arrested using a low-dose treatment of Mitomycin-C, and have no apparent toxicity or change in cellular signal transduction. Both RAR alpha DA cells and RAR alpha-UAS-bla HEK 293T cells have been tested for assay performance using variable assay conditions, including DMSO concentration, cell number, stimulation time, substrate loading time and have been validated for Z' and EC50 concentrations of all-trans retinoic acid. Additional testing data using alternate stimuli are also available.

GeneBLAzer™ EDG3-Gα15 HEK 293T DA Assay Kit

This cell line is available as dividing cells by requesting a quote for K1234 from dividingcells@invitrogen.com

GeneBLAzer® EDG3-Gα15 HEK 293T DA (Division Arrested) cells and EDG3-Gα15-NFAT-bla HEK 293T cells contain the human EDG3 receptor stably integrated in the GeneBLAzer® Gα15-NFAT-bla HEK 293T cell line. GeneBLAzer® Gα15-NFAT-bla HEK 293T cells (#K1212) contain a beta-lactamase reporter gene under control of the Nuclear Factor of Activated T-cells (NFAT) response element, and stably express the promiscuous G-protein, Gα15. Division Arrested (DA) cells are available in two configurations: an Assay Kit (which includes cells and sufficient substrate to analyze 1 x 384-well plate), and a tube of cells sufficient to analyze 10 x 384-well plates.

DA cells are irreversibly division arrested using a low-dose treatment of Mitomycin-C, and have no apparent toxicity or change in cellular signal transduction. Both EDG3-Gα15 HEK 293T DA cells and EDG3-Gα15-NFAT-bla HEK 293T cells are functionally validated for Z’-factor and EC50 concentrations of Sphingosine-1-phosphate (S1P), (Figure 1). In addition, EDG3-Gα15-NFAT-bla HEK 293T cells have been tested for assay performance under variable conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time (data available upon request). Additional testing data using alternate stimuli are also available.

EDG-3(Endothelial-differentiation-gene-3)/S1P-3(Sphingosine-1-Phosphate-3) is a Gq/Gi/Go/G13/G12 coupled GPCR. EDG-3 has been shown to be responsible for bradycardia that has been induced in clinical trials as a side effect of the immunosuppressive drug FTY720 that targets EDG-1. EDG-3 has also been shown to induce vasodilation in response to sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and lysosulfatide (LSF). The EDG-3 Receptor has also been shown to play a cooperative or redundant role in angiogenesis with EDG-1

GeneBLAzer™ PTGER2-NFAT-bla CHO-K1 Cells

For academic pricing please login or contact us at discoverysciences@invitrogen.com.

GeneBLAzer® PTGER2-NFAT-bla CHO-K1 cells contain the human Prostaglandin E Receptor 2 (PTGER2), (Accession # NM_000956.2) stably integrated into the CellSensor® NFAT-bla CHO-K1 cell line. CellSensor® NFAT-bla CHO-K1 cells (Cat. no. K1534) contain a beta-lactamase reporter gene under control of the NFAT Response Element.

GeneBLAzer® PTGER2-NFAT-bla CHO-K1 cells have been tested for assay performance under variable conditions. These data are found in the Validation & Assay Performance Summary.