Targets / Signaling Pathways
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Signaling Pathway Product Solutions

Interrogate a Variety of Popular Target-Specific Pathways with Cell-Based Assays

Cellular pathway signaling constitutes key mechanisms that transfer biological information within cells for intracellular responses crucial in many physiological processes including cell proliferation, differentiation, metabolic control, and apoptosis. Cell signaling pathways are activated when ligands (e.g., cytokines, growth factors, or hormones) bind to specific receptors on the cell surface. Detecting specific stimuli via intracellular signal pathways becomes paramount in interrogating the mechanism of action of the target ligand/receptor interaction for advancing drug discovery and development in many pathophysiologic and pharmacologic mechanisms.

 

Eurofins DiscoverX® offers a comprehensive collection of cell-based pathway indicator assays designed to detect activation or inhibition of complex signal transduction pathways in response to a small molecule or biologic. The assays are based on the established PathHunter® technology, and feature indicator cell lines used in pathway assays that enable you to measure distinct events within a variety of pathways associated with immune response, compound toxicity, cholesterol metabolism, antioxidant function, DNA damage, and more.

 

Combined with a fast and simple chemiluminescent detection, these signaling pathway assays enable the generation of cellular pathway selectivity profiles and obtain a comprehensive understanding of different compounds or biologics for receptor-proximal or -distal events without relying on complex phenotypic screens.

 

Advantages of Signaling Pathway Assays

  • Homogeneous Assays – Simple, no wash HTS-friendly protocols with a sensitive chemiluminescent read-out
  • Multiple Assay Types – Binding and functional (direct and reporter-based) cell-based assay with no need for antibodies, fixing, or imaging
  • Fast, Superior Results – Rapid assay with short compound incubation times, high signal-to-background, and no false positives
  • High Specificity – Whole cell, non-ELISA assays with increased target specificity

Related Topics

Custom Products

  • Custom Capabilities – Custom cell lines, assays, and protein development capabilities optimized to your requirements

Apoptosis Signaling Pathway Products

Cancer cells are known to over-express a number of anti-apoptotic proteins that belong to the BCL2 family on the surface of the mitochondria. These proteins interact with pro-apoptotic proteins and regulate the intrinsic apoptotic pathway by controlling the mitochondrial membrane permeability and release of the pro-apoptotic factor cytochrome c that initiates activation of the caspase cascade and apoptosis. Eurofins DiscoverX offers novel, direct, cell-based assays to study these apoptotic players. These apoptosis signaling pathway assays are based on the InCELL Hunter™ target engagement destabilization assay. The portfolio covers pre-validated cell lines and assay-ready kits that allow you to screen for protein interaction inhibitors (small molecules or siRNA) in an HTS-friendly and simple assay format.

Cell Lines

Target Description Cat. No.
Bcl2-Bax InCELL Hunter™ U2OS Bcl2-Bax Protein Binding Cell Line 96-0010C3
Bcl2-Bim InCELL Hunter™ U2OS Bcl2-Bim Protein Binding Cell Line 96-0008C3
Bcl2L1-Bax InCELL Hunter™ U2OS Bcl2L1-Bax Protein Binding Cell Line 96-0009C3
Bcl2L1-Bim InCELL Hunter™ U2OS Bcl2L1-Bim Protein Binding Cell Line 96-0037C3

Assay Ready Kits

Target Description Cat. No.
Bcl2L1-Bax InCELL Hunter™ eXpress Bcl2L1-Bax Protein Binding Assay 96-0009E3CP16L
Bcl2L1-Bax InCELL Hunter™ eXpress Bcl2L1-Bax Protein Binding Assay 96-0009E3CP16M
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TORC (CRTC) Signaling Pathway Products

cAMP-regulated transcriptional co-activators TORC1 (CRTC1) and TORC2 (CRTC2) are known to regulate the cAMP-responsive element-binding (CREB) protein. Rising intracellular cAMP levels lead to de-phosphorylation of CRTC1 and CRTC2 that leads to the translocation of CRTC to the nucleus. CRTC translocation is known to be regulated by AMPK (5'-AMP activated kinase), PKA, and SIK. TORC2 and AMPK are potential therapeutic targets for Type II Diabetes. Eurofins DiscoverX offers novel direct, functional cellular assays in the form of PathHunter cell lines and assay-ready kits for TORC1 (CRTC1) and TORC2 (CRTC2) translocation. These products enable measurement of compound effects on CRTC1 and CRTC2 translocation or for screening and profiling small molecules, siRNA, or inhibitors of upstream targets AMPK, S1K, or CRTC.

Cell Lines

Target Description Cat. No.
TORC1 (CRTC1) PathHunter® U2OS TORC1 Nuclear Translocation Cell Line 93-0833C3
TORC2 (CRTC2) PathHunter® U2OS TORC2 Nuclear Translocation Cell Line 93-0813C3
TORC3 (CRTC3) PathHunter® U2OS TORC3 Nuclear Translocation Cell Line 93-1120C3

Assay Ready Kits

Target Description Cat. No.
TORC1 (CRTC1) PathHunter® eXpress TORC1 Nuclear Translocation Assay 93-0833E3CP19L
TORC1 (CRTC1) PathHunter® eXpress TORC1 Nuclear Translocation Assay 93-0833E3CP19M
TORC2 (CRTC2) PathHunter® eXpress TORC2 Nuclear Translocation Assay 93-0813E3CP19L
TORC2 (CRTC2) PathHunter® eXpress TORC2 Nuclear Translocation Assay 93-0813E3CP19M
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DNA Damage Signaling Pathway Cell Line

DNA damage results in cell cycle arrest, apoptosis, and the stabilization and repair of the cellular genome. The DNA damage PathHunter assay includes downstream targets such as ATM, ATR, CDC25A, Chk1, Chk2, Caspase 3, and p21, and signals through p53-dependent and -independent processes. Eurofins DiscoverX offers a novel direct, functional cellular assay for predictive toxicology that is reliable, easy to run, user-friendly, and compatible with 384 well protocols. This assay in the form of a cell line allows you to obtain the effect of your early stage compound on key cellular pathways such as DNA damage (p53) or ER stress. The cell line provides a scope for screening targets upstream of the p53 pathway or for studying compound toxicity profiles (genotoxic markers).

Cell Line

Target Description Cat. No.
p53 PathHunter® U2OS p53 Nuclear Translocation Cell Line 93-0757C3
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NF-κB and IκB Signaling Pathways Products

Nuclear factor kappa B (NF-κB) belongs to a family of transcription factors that play pivotal roles in inflammatory responses and immunological reactions. The NF-κB pathway can be activated by a variety of stimuli, including TNF-α (tumor necrosis factor alpha), interleukin 1 (IL-1), T and B cell mitogens, bacterial lipopolysaccharide (LPS), and viral proteins. Eurofins DiscoverX offers simple one-step degradation, reporter, and nuclear translocation assays to study the NF-κB pathway. These fully optimized, target-validated cell lines provide you convenience, relevant hits, and guaranteed performance for screening, profiling, or research applications, including monitoring the degradation of IκB or screening inhibitors for TNFα signaling or IκB kinases.

Cell Lines

Target Description Cat. No.
RANK NF-κB PathHunter® U2OS RANK NF-κB Reporter Cell Line 93-1156C3
NF-κB PathHunter® U2OS NF-κB Reporter Cell Line 93-1157C3
IκB PathHunter® HEK 293 IκB Degradation Cell Line 93-0538C1
IκB PathHunter® THP-1 IκB Degradation Cell Line 93-0538C14
IκB PathHunter® A549 IκB Degradation Cell Line 93-0538C15
IκB PathHunter® U2OS IκB Degradation Cell Line 93-0538C3
IκB PathHunter® Jurkat IκB Degradation Cell Line 93-1121C19
RANK-IκB PathHunter® U2OS RANK-IκB Functional Assay 93-0994C3
RELA PathHunter® HEK 293 RELA Nuclear Translocation Cell Line 93-0907C1

Assay Ready Kits

Target Description Cat. No.
IκB PathHunter® Adalimumab Bioassay Kit 93-0538B15-00131
IκB PathHunter® Adalimumab Bioassay Kit 93-0538B15-00132
IκB PathHunter® eXpress HEK 293 IκB Degradation Assay 93-0538E1CP7L
IκB PathHunter® eXpress HEK 293 IκB Degradation Assay 93-0538E1CP7M
RELA PathHunter® eXpress HEK 293 RELA Nuclear Translocation Assay 93-0907E1CP0L
RELA PathHunter® eXpress HEK 293 RELA Nuclear Translocation Assay 93-0907E1CP0M
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NRF2 Signaling Pathways Products

NRF2 [Nuclear factor (erythroid-derived 2)-like 2] and its downstream target genes are the primary cellular defense against the cytotoxic effects of oxidative stress. Cell damage due to free radicals from oxidative stress contributes to diseases such as arteriosclerosis, stroke, asthma, cardiac infarctions, Alzheimer's disease, Parkinson's disease, and cancer, making the NRF2 pathway a high-profile therapeutic target.

Cell Lines

Target Description Cat. No.
Keap1-NRF2 PathHunter® U2OS Keap1-NRF2 Nuclear Translocation Cell Line 93-0821C3

Assay Ready Kits

Target Description Cat. No.
Keap1-NRF2 PathHunter® eXpress Keap1-NRF2 Nuclear Translocation Assay 93-0821E3CP0L
Keap1-NRF2 PathHunter® eXpress Keap1-NRF2 Nuclear Translocation Assay 93-0821E3CP0M
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PI3K Signaling Pathways Products

PI3K/AKT signaling regulates cellular processes such as proliferation, growth, survival, apoptosis, and the up regulation of hypoxia-related proteins. Screen for inhibitors to PI3 kinase and AKT and analyze receptor activation, phosphorylation, or downstream FOXO translocation in an HTS-friendly, cell-based format without the need for antibodies

Cell Lines

Target Description Cat. No.
PI3 PathHunter® U2OS FOXO3 Nuclear Translocation Cell Line 93-0876C3

Assay Ready KitsPI3

Target Description Cat. No.
PI3 PathHunter® eXpress FOXO3 Nuclear Translocation Assay 93-0876E3CP5L
PI3 PathHunter® eXpress FOXO3 Nuclear Translocation Assay 93-0876E3CP5M
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SREBP Signaling Pathways Products

Membrane-bound transcription factors, sterol regulatory element binding protein-1 and -2 (SREBP-1 and SREBP-2), mediate cholesterol and fatty acids homeostasis. SREBP-2 monitors cellular cholesterol levels and responds to low levels of cholesterol by the transcription of genes for HMG-CoA Reductase and other enzymes involved in the cholesterol synthesis pathway. Recent studies implicate SREBP-2 in hypercholesterolemia, coronary heart disease, obesity, autophagy, and Alzheimer's disease. Eurofins DiscoverX offers novel direct, functional cell-based assays for SREBP2 translocation capable of screening or profiling the SREBP cholesterol sensing pathway inhibitors and measuring the effect of compounds on SREBP2 translocation.

Cell Lines

Target Description Cat. No.
SREBP2 PathHunter® U2OS SREBP2 Nuclear Translocation Cell Line 93-0830C3

Assay Ready Kits

Target Description Cat. No.
SREBP2 PathHunter® eXpress SREBP2 Nuclear Translocation Assay 93-0830E3CP5L
SREBP2 PathHunter® eXpress SREBP2 Nuclear Translocation Assay 93-0830E3CP5M
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TGFβR Signaling Pathways Products

TGFβ (Transforming Growth Factor beta) is a small secreted polypeptide that signals through the type II serine/threonine kinase dimeric receptor (TGFβR2) that recruits and phosphorylates the type I dimeric receptor (TGFβR1). TGFβR1 phosphorylates and activates the SMAD transcription factors that regulate genes involved in cell proliferation, differentiation, apoptosis, and growth. Many advanced stage cancers are known to over-express TGFβ and TGFβR, promoting aggressive tumor formation. Inhibiting the TGFβ signaling pathway is a key therapeutic strategy in treating cancer. Enable your therapeutic programs by discovering small molecule inhibitors or biologics to TGFβR by using a robust and simple PathHunter TGFβR1/TGFβR2 Dimerization Assay. Analyze small molecule and biologics (antibody) receptor dimerization inhibitors and study compound toxicity profiles.

Cell Lines

Target Description Cat. No.
TGFβR1/TGFβR2 PathHunter® U2OS TGFBR1/TGFBR2 Dimerization Cell Line 93-0889C3
TGFβR1A/ACVR2A PathHunter® U2OS TGFBR1A/ACVR2A Dimerization Cell Line 93-1012C3
TGFβR1/ACVR2B PathHunter® U2OS TGFBR1/ACVR2B Dimerization Cell Line 93-1046C3
TGFβR1/TGFβR2/ENG PathHunter® U2OS TGFBR1/TGFBR2/ENG Dimerization Cell Line 93-1122C3

Assay Ready Kits

Target Description Cat. No.
TGFβR1/TGFβR2 PathHunter® eXpress TGFBR1/TGFBR2 Dimerization Assay 93-0889E3CP5L
TGFβR1/TGFβR2 PathHunter® eXpress TGFBR1/TGFBR2 Dimerization Assay 93-0889E3CP5M
TGFβR1/ACVR2 PathHunter® eXpress TGFBR1/ACVR2 Dimerization Assay 93-1012E3CP5L
TGFβR1/ACVR2 PathHunter® eXpress TGFBR1/ACVR2 Dimerization Assay 93-1012E3CP5M
TGFβR1/ACVR2B PathHunter® eXpress TGFBR1/ACVR2B Dimerization Assay 93-1046E3CP5L
TGFβR1/ACVR2B PathHunter® eXpress TGFBR1/ACVR2B Dimerization Assay 93-1046E3CP5M
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Wnt-frizzled Signaling Pathways Products

Wnt signaling plays a pivotal role in development, and it is implicated in a variety of disease states ranging from cancer to kidney and reproductive tract defects. Wnt ligands are associated with both β-Catenin dependent (canonical) and β-Catenin independent pathways. Analyze small molecules, siRNA, or functional antibodies that activate the frizzled (Fz) GPCR receptor and inhibit upstream GSK3β.

Cell Lines

Target Description Cat. No.
Wnt-FzGSK3β PathHunter® U2OS β-Catenin Nuclear Translocation Cell Line 93-0743C3

Assay Ready Kits

Target Description Cat. No.
Wnt-FzGSK3β PathHunter® eXpress β-Catenin Nuclear Translocation Assay 93-0743E3CP22L
Wnt-FzGSK3β PathHunter® eXpress β-Catenin Nuclear Translocation Assay 93-0743E3CP22M
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Signaling pathway assays are based on Eurofins DiscoverX’s Enzyme Fragment Complementation (EFC) Assay platform. These assays are available as cell lines and assay-ready kits to enable you to measure distinct events within various pathways associated with immune response, compound toxicity, cholesterol metabolism, antioxidant function, DNA damage, ER stress, and more.

 

Apoptosis Signaling Pathway Assay Principle

Apoptosis Signaling Pathway Assay Principle

These apoptosis signaling pathway assays are based on the InCELL Hunter™ target engagement, destabilization assay. Cell lines are engineered to co-express an untagged target protein and an interacting partner protein fused with ePL (enhanced ProLabel®; the small EFC enzyme donor fragment of β-galactosidase (β-gal)). In the absence of an inhibitor compound and addition of the large EFC enzyme acceptor (EA) β-gal fragment, the untagged target and ePL-tagged partner protein interact, resulting in stable steady-state levels of ePL-tagged partner protein. Addition of inhibitors that bind the target protein and disrupt the target-partner protein interaction results in ePL-tagged partner protein degradation. The abundance of ePL-tagged partner protein in the presence of a substrate that is hydrolyzed is measured by chemiluminescent detection indicated by complementation of the ePL and EA fragments to make an active β-gal enzyme. The greater the signal corresponds to less compound-target engagement in the cell.

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TORC (CRTC) Signaling Pathway Assay Principle

TORC (CRTC) Signaling Pathway Assay Principle

PathHunter TORC1 or TORC2 assays measure TORC1 or TORC2 translocation from the cytosol to the nucleus and subsequent binding to TAZ1 protein. The cell lines have been engineered to express the two complementing EFC fragments of the β-gal enzyme within different cellular compartments. Full length human CRTC1 (TORC1) gene or CRTC2 (TORC2) is N-terminally tagged with ProLink™ (PK; the small enzyme donor of β-gal). TAZ1 (transcriptional adaptor zinc-binding domain) is C-terminally tagged with EA (the large enzyme acceptor of β-gal) and is localized in the cell nucleus. Phosphorylated TORC does not translocate, but the non-phosphorylated TORC translocates into the nucleus and binds to TAZ1 protein. Activation of the pathway initiates TORC1 translocation to the nucleus that forces EFC complementation of the two β-gal enzyme fragments (PK and EA). This action results in the formation of fully active β-gal enzyme, which can hydrolyze a substrate to generate a chemiluminescent signal that can be easily measured on any standard luminometer.

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DNA Damage Signaling Pathway Assay Principle

DNA Damage Signaling Pathway Assay Principle

The DNA damage PathHunter assay monitors the translocation of p53 from the cytosol to the nucleus. The cell line has been engineered to express the small EFC enzyme donor fragment PL (ProLabel) of β-gal attached to p53 and the larger EFC enzyme acceptor fragment (EA) localized in the nucleus. Activation of the DNA damage pathway initiates protein translocation of p53 to the nucleus that forces complementation of the two β-gal enzyme fragments (PL and EA). This action results in the formation of fully complemented β-gal enzyme, the activity of which is measured using a chemiluminescent substrate. The signal can be read on any standard luminometer.

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NF-κB and IκB Signaling Pathways Assay Principles

NF-κB and IκB Signaling Pathways Assay Principles

NF-κB and IκB signaling pathways assay principles. A. NF-κB signaling reporter assay construct regulates PathHunter EFC reporter expression via upstream NF-κB response elements controlled by NF-κB transcription factor binding. Upstream signaling events result in NF-κB nuclear translocation and subsequent response-element binding. The signaling activity is measured by monitoring the abundance of a reporter protein, which is tagged with the small EFC enzyme donor PL (ProLabel) fragment. Upon addition of the larger EFC enzyme acceptor (EA) fragment, complementation of the two β-gal enzyme fragments (PL and EA) occurs creating an active β-gal enzyme, which is capable of substrate hydrolysis and production of a chemiluminescent signal. B. PathHunter IκB degradation and RelA translocation assays are direct functional assays. In the IκB degradation assay (left pathway), IκB is tagged with the small EFC enzyme donor PL fragment. The assay measures human IκB degradation in response to activation of the NF-kB pathway. In the presence of TNFα or other compounds, cells signal through downstream IκB proteins and the IκB protein is degraded in response to compound stimulation. Upon addition of EA, complementation of the two β-gal enzyme fragments occurs creating an active β-gal enzyme, which is capable of substrate hydrolysis and production of a chemiluminescent signal. In the RelA (NF-kB p65) nuclear translocation assay (right pathway), p65 (RelA) is tagged with the small EFC enzyme donor Pk (ProLink) fragment and a nuclear protein (TAZ1) is tagged with EA. The assay measures RelA translocation to the nucleus in response to activation of the NF-κB pathway. In the presence of TNFα or other compounds, cells signal through downstream IκB proteins and causes RelA to translocate to the nucleus where complementation of the two β-gal enzyme fragments occurs creating an active β-gal enzyme, which is capable of substrate hydrolysis and production of a chemiluminescent signal.

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NRF2 Signaling Pathways Assay Principle

NRF2 Signaling Pathways Assay Principle

The PathHunter KEAP1-NRF2 Translocation Assay incorporates the small EFC enzyme donor PK (Prolink) fragment fused to the NRF2 protein in a recombinant cell line, while the larger EFC enzyme acceptor (EA) fragment is anchored in the nucleus. Activation of the Keap1-NRF2 protein complex results in NRF2 stabilization and translocation to the nucleus, resulting in PK-EA complementation and formation of a functional β-gal enzyme. Enzyme activity is then quantitatively detected using the β-gal substrate, which is hydrolyzed to produce a chemiluminescent signal.

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PI3K Signaling Pathways Assay Principle

PI3K Signaling Pathways Assay Principle

The PathHunter FOXO3 Nuclear Translocation Assay incorporates the small EFC enzyme donor PK (Prolink) fragment fused to the FOXO-3 protein in a recombinant cell line, while the larger EFC enzyme acceptor (EA) fragment is anchored in the nucleus. Activation of the pathway initiates FOXO-3 translocation to the nucleus forces the complementation of PK-EA fragments and the formation of a functional β-gal enzyme. Enzyme activity is then quantitatively detected using the β-gal substrate that is hydrolyzed to produce a chemiluminescent signal.

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SREBP Signaling Pathways Assay Principle

SREBP Signaling Pathways Assay Principle

The PathHunter SREBP2 Nuclear Translocation Assay incorporates the small EFC enzyme donor PK (Prolink) fragment fused to the SREBP2 protein in a recombinant cell line, while the larger EFC enzyme acceptor (EA) fragment is anchored in the nucleus. Low cholesterol activates PK-SREB2/Scap protein complex that blebs off from endoplasmic reticulum and traverses to Golgi where it is cleaved by S1P. The SREBP2-PK protein translocates to nucleus forcing complementation of the PK-EA fragments and formation of a functional β-gal enzyme. Enzyme activity is then quantitatively detected using the β-gal substrate, which is hydrolyzed to produce a chemiluminescent signal.

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TGFβR Signaling Pathways Assay Principle

TGFβR Signaling Pathways Assay Principle

The PathHunter dimerization assays incorporate the small EFC enzyme donor PK (Prolink) fragment fused to one receptor, TGFβR1, and the larger EFC enzyme acceptor (EA) fragment fused to the other receptor, TGFβR2. Agonist binding to TGFβR2 induces its heterodimerization with TGFβR1 forcing the complementation of the PK with EA fragment resulting in a fully complemented active β-gal enzyme. Enzyme activity is then quantitatively detected using the β-gal substrate, which is hydrolyzed to produce a chemiluminescent signal.

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Wnt-frizzled Signaling Pathways Assay Principle

Wnt-Frizzled Signaling Pathways Assay Principle

The PathHunter Wnt-frizzled assay incorporates the small EFC enzyme donor PL (ProLabel) fragment fused to the β-catenin protein in a recombinant cell line, while the larger EFC enzyme acceptor (EA) fragment is anchored in the nucleus. Activation of the Wnt-frizzled signaling pathway initiates β-catenin translocation to the nucleus forcing complementation of the PK-EA fragments and formation of a functional β-gal enzyme. Enzyme activity is then quantitatively detected using the β-gal substrate, which is hydrolyzed to produce a chemiluminescent signal.

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Access Products by Target Families

Bcl2-Bax p53 RELA TGFBR1A/ACVR2A
Bcl2L1-Bax RANK NF-kB Keap1-NRF2 TGFBR1/ACVR2B
TORC1 (CRTC1) NF-kB PI3 TGFBR1/TGFBR2/ENG
TORC2 (CRTC2) IkB SREBP2 β-Catenin
TORC3 (CRTC3) RANK IkB TGFBR1/TGFBR2 FOXO3