Binary assay

Reporter assay

The simplest MAPPIT read-out is a luciferase reporter assay. A plasmid encoding firefly luciferase driven by the STAT3-responsive rPAP1 promoter is co-transfected in HEK293-T cells together with plasmids encoding the chimeric bait receptor and the prey. After stimulation with the appropriate ligand (e.g. epo, leptin or GM-CSF, depending on the choice of the extracellular part of the bait receptor), cells are lysed and luminescence is measured after adding the luciferin substrate.

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STAT3 activation assay

In the signalling cascade following bait-prey interaction and ligand addition, one of the very first events is tyrosine phosphorylation of STAT3. We are developing a sensitive phospho-STAT3 ELISA assay with a read-out within 15 minutes after receptor stimulation. This can be useful when toxicity of the tested compounds is suspected in ReverseMAPPIT or MASPIT experiments, or when interference with the reporter assay is suspected.