In the three-hybrid mode, MASPIT, non-protein organic molecules can be displayed as bait through use of chemical dimerizers. A signalling-deficient receptor is fused to DHFR (dihydrofolate reductase), which is able to capture with very high affinity MTX (methotrexate) fusion compounds. These consist of the organic molecule that serves as bait tethered to MTX via a PEG (polyethylene glycol) linker. Binding of a protein prey to the MTX fusion compound complements the STAT3 signalling cascade initiated upon ligand binding. Binary assays can be performed to test specific compound-protein interactions, but MASPIT also allows screening for novel protein targets of a certain organic molecule, either using the FACS-based cDNA library screening protocol or the array-based procedure.

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