[DOT 2020] New PathHunter Reporter Assays: Analysis of PD-1 Checkpoint Receptor and NFAT Signaling Pathways

[DOT 2020] New PathHunter Reporter Assays: Analysis of PD-1 Checkpoint Receptor and NFAT Signaling Pathways
File Name/Number:
DOT 2020

Year:
2012


Reporter genes are a well-established method used to develop cell-based assays for testing drugs that inhibit targets involved in specific signaling pathways. Here, we introduce new PathHunter® Signaling Pathway Reporter Assays that utilize the industry-validated Enzyme Fragment Complementation (EFC) technology to detect reporter gene activity. Reporter cells with endogenous or heterologously introduced target receptors are used to readout signaling pathway activation, resulting in transcriptional activation of a reporter gene encoding a protein tagged with a small enzyme fragment. Reporter gene activity is measured by the addition of lysis buffer, luminescent enzyme substrate and the complementary larger enzyme fragment in an easy-to-use format. For example, an NF-κB reporter assay was developed and validated with CD40L to measure endogenous CD40 activation in U2OS cells. Another assay developed is the PathHunter Jurkat NFAT Pathway Reporter cell line, which measures T-cell activation but has been modified further to build an assay for testing therapeutic compounds inhibiting the inhibitory PD-1 checkpoint receptor effects on T-cell activation. The PathHunter Jurkat PD-1 Pathway Reporter Assay provides an alternative means of investigating the PD-1 signaling pathway that is complementary to our PathHunter Jurkat PD-1 Signaling Assay, which reads out effects on more proximal events in the PD-1 pathway independent of T-cell receptor activation.

Listen to a short recording of the poster by Jennifer Lin-Jones, Ph.D.