Signaling Pathway Reporter Assays

A Simple, Orthogonal Screening Tool for Understanding Therapeutic MOAs

Cell-based reporter assays are well established and used to evaluate the cellular impact of therapeutics targeting a variety of proteins in signaling pathways. The PathHunter® Signaling Pathway Reporter products are easy-to-use, functional cell-based assays for screening small molecule or biologic drugs and quantifying the activation and inhibition of various signaling pathways. These assays provide a downstream (transcription/translational) read-out that is complementary to receptor proximal-based assay read-outs to ultimately gain a comprehensive understanding of your drug molecule’s MOA targeting TNFα, PD-1, IL-6, CD40, RANK and other receptors.


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Advantages Of Signaling Pathway Reporter Assays

PathHunter Signaling Pathway Reporter Assays provide a screening tool for developing drugs that target diverse signaling pathways. These simple, robust and sensitive cell-based reporter assays offer an orthogonal method to receptor proximal read-out assays to quantify the activation and inhibition of several different signaling pathways (e.g. NF-kB, NFAT and STAT3).

  • Complete Understanding – Evaluate both receptor-proximal and receptor-distal responses for a comprehensive understanding of the drug molecule’s MOA
  • Versatile – Suitable for screening of agonists or antagonists and cell lines can be used to build assays specific for additional targets utilizing a pathway
  • Simple Protocol, Fast Results — Easy-to-run, rapid homogeneous protocol amenable to implementation in multiple labs and high-throughput format for increased efficiency
  • Biologically-Relevant — MOA-reflective, functional assays for monitoring diverse signaling and testing of small molecule or biologic drugs

Key Resources

PathHunter Signaling Reporter assays are available as stable cell lines used with PathHunter detection reagents for the assessment of diverse signaling pathways

Cell Lines

Target Description Cat. No.
NFAT PathHunter® Jurkat NFAT Pathway Reporter Cell Line 93-1141C19
NFkB PathHunter® U2OS NF-KB Reporter Cell Line 93-1157C3
RANK-NFkB PathHunter® U2OS RANK-NF-KB Reporter Cell Line 93-1156C3
STAT3 PathHunter® HepG2 STAT3 Pathway Reporter Cell Line 93-1144C44
PD-1-NFAT PathHunter® Jurkat PD1-NFAT Pathway Reporter Cell Line inquire
NF-κB PathHunter® HEK293 NF-κB Pathway Reporter Cell Line inquire
CD27-NF-κB PathHunter® HEK293 CD27-NF-κB Pathway Reporter Cell Line inquire
TREM1-NFAT PathHunter® Jurkat TREM1-NFAT Pathway Reporter Cell Line inquire
IL11R-STAT3 PathHunter® HepG2 IL11R-STAT3 Pathway Reporter Cell Line inquire

The PathHunter Signaling Pathway Reporter Assays are stable clonal cell lines based on the enzyme fragment complementation (EFC) technology and used for monitoring the activation or inhibition of various signaling pathways. Agonist ligands bind to distinct receptors and trigger specific signaling pathways. Transcription factors are then activated, followed by their nuclear translocation. Binding of these activated transcription factors to specific promoter elements regulates downstream gene transcription, leading to protein expression.

Assay Principle


PathHunter Signaling Pathway Reporter assays detect target pathway signaling through the activation of endogenous receptors or receptors introduced into cells with the reporter gene construct. Ligand-mediated stimulation of these receptors initiates pathway signaling and subsequent activation of transcription factors, which bind to a regulatory transcriptional element controlling reporter gene expression.  In this assay, the activated signaling pathway drives the expression of the reporter protein tagged with the small enhanced ProLabel (ePL) β-galactosidase enzyme donor fragment.  Reporter activity is measured by lysing reporter pathway cells with a detection reagent containing the complementary enzyme acceptor (EA) fragment and luminescent enzyme substrate.  The enzyme activity is then detected as a result of EFC.


Quantify the Activation and Inhibition of Signaling Pathways

PathHunter signaling reporter assays for endogenous or heterologously-expressed target receptors.  A. The PathHunter U2OS NF-κB Pathway Reporter Assay detects CD40L-mediated activation of endogenous CD40 receptors. B. The PathHunter U2OS RANK-NFκB Pathway Reporter Assay was created by RANK gene transfer into PathHunter U2OS NFκB Pathway Reporter cells to generate a sensitive assay for RANK activation by RANKL. C. The PathHunter U2OS RANK-NFκB Pathway Reporter Assay was used to analyze antagonist activity of the therapeutic RANKL inhibitor, Prolia® (registered trademark of Amgen) and shows a robust and sensitive response with a large signal-to-background ratio. [Prolia + 10ng/mL RANKL exhibited an IC50 of 23 ng/mL and an S/B ratio of 25.9, while RANKL alone resulted in an EC50 of 7.7 ng/mL and an S/B ratio of 29.1.]

Develop Potent Agonist and Antagonist Therapeutics


Develop therapeutics targeting receptors with the various signaling pathway reporter assays. The PathHunter HepG2 STAT3 Pathway Reporter Assay was used to detect the potent antagonist therapeutic, Tocilizumab (Actemra®, registered trademark of Genentech), which has been approved for clinical trials to treat patients with severe Covid-19 pneumonia.

Monitor T Cell Activation

The PathHunter Jurkat NFAT Pathway Reporter Assay monitors T-cell activation signaling through T-cell receptors and results in changes in NFAT-regulated reporter gene expression. Jurkat NFAT reporter cells were co-cultured with increasing numbers of U2OS cells expressing a molecule that activates Jurkat T-cell receptors to initiate T-cell activation. The ePL-tagged reporter protein is expressed when a sufficient number of U2OS T-cell activator cells engage endogenous Jurkat T-cell receptors to activate the pathway and drive NFAT-dependent transcription and translation of the ePL-tagged reporter protein.

Measure Sensitive Responses, from Either Distal or Proximal Events


Comparison of antagonist testing results from two different PathHunter PD-1 Assays. A. The PathHunter PD-1 Pathway Reporter Assay was tested in a dose-response format with an Anti-PD-1 Antibody. Reporter assay cells co-cultured with the PathHunter U2OS PD-L1/TCR activator cell line results in increased reporter expression due to blocking PD-1 inhibition of TCR activation, resulting in increased NFAT-regulated gene expression. The reporter assay readout measures downstream effects of PD-1 receptor signaling by resulting in NFAT-regulated reporter protein expression. Conversely, B. represents results from PathHunter Jurkat PD-1 SHP2 Signaling Assay antagonist testing. An Anti-PD-1 Antibody was used to block PD‑1 activation mediated by PathHunter U2OS Ligand Cell Line co-culture. This assay measures proximal PD-1 signaling events independent of TCR activation. Both assays are robust and measure inhibition with sensitive responses, from either distal or proximal events.