HTRF® Total and Phospho-YAP (Ser127) Cellular Assays
The YAP protein is a co-transcription factor increasing the expression of genes involved in cell proliferation and repressing the expression of genes important for cell differentiation and apoptosis. It is regulated by several inputs, like the Hippo pathway, through the action of kinases that phosphorylate YAP on several residues. The level of phosphorylation of the residues serine 127 (S127) of YAP is generally assessed in cellular models, native tissues, and organs, as a marker of YAP activity and location, and is regulated by numerous partners. This phosphorylation event is classically detected using a western blot technical approach. Here, we describe a novel approach to detect both the relative amount of total YAP (T-YAP assay) and the phosphorylation of the residue S127 of YAP (S127-P-YAP assay) using a HTRF®-based method. This easy-to-run method can easily be miniaturized and allows for a high-throughput analysis in 96/384-well plate format, requiring less cellular material and being more rapid than other approaches.
Key wordsYAP phosphorylation YAP-S127-phosphorylation-directed antibodies Antibody-based assay Resonance energy transfer HTRF®
The work described was made possible through Cisbio Bioassays and the technological pharmacology facilities ARPEGE (Pharmacology Screening Interactome) of Biocampus (IGF). This work has been supported by CisBio (cooperative research team Eidos, CNRS N°039293/CBB DRD-09-04 avenant 4), the Labex EpiGenMed, an “Investissements d’avenir” program (ANR-10-LABX-12-01), the Fondation pour la Recherche Médicale (DEQ20170336747), CNRS, Inserm, and the University of Montpellier.
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