HTRF® total and phospho-YAP(Ser127) cellular assays

Diana Zindel (1) , Claire Vol (1) , Odile Lecha (1) , Isabelle Bourquignon (1) , Merve Bilgic (1) , Marion Vereecke (1) , Fabienne Charrier-Savournin (2) , Maité Romier (2) , Eric Trinquet (2) , Jean-Philippe Pin (1) , Julie Pannequin (1) , Thomas Roux (2) , Elodie Dupuis (2) , Laurent Prézeau (1)

1 IGF - Institut de Génomique Fonctionnelle
2 Innovation Management / CbB - Cisbio Bioassays [Codolet, France]

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.

Mots clés: YAP-S127-phosphorylation-directed antibodies; Resonance energy transfer; YAP phosphorylation; HTRF®; Antibody-based assay

Domaines: Neurosciences [q-bio.NC]; Biotechnologies

Diana Zindel, Claire Vol, Odile Lecha, Isabelle Bourquignon, Merve Bilgic, et al.. HTRF® total and phospho-YAP(Ser127) cellular assays. The Hippo pathway: Methods and Protocols, 1893, pp.153-166, 2019, Methods in Molecular Biology, ⟨10.1007/978-1-4939-8910-2_13⟩. ⟨hal-02396286⟩