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Automation of Homogeneous Proximity assays for Detection of Erk1/2 or Smad3 PhosphorylationDownload
February 13, 2015
Authors: P. Brescia, P. Banks and C. Foster, BioTek Instruments, Inc.
Cell signaling is generally initiated by cell surface receptor ligand binding events resulting in activation of serine/threonine receptor kinases. The concomitant phosphorylation of cytoplasmic signaling molecules begins a signaling cascade. The transforming growth factor-β (TGF-β) superfamily consist of a range of proteins involved in a wide array of biological processes such as cell growth, differentiation, and development. TGF-β signaling occurs with the cell through the Smad family of transcriptional activators. Smad 2 and Smad 3, present in the TGF-β/activin Smad pathway, are wellstudied phosphopuoteins for their potential as drug targets for disorders such as cardiovascular, musculoskeletal, fibrosis and cancer. ERK 1 and 2 (extracellular signal-regulated kinase 1 and 2) have been shown to be regulated by both receptor tyrosine kinase receptors (RTKs) and G protein-coupled receptors (GPCRs) activation. Erk1/2 have been shown to have a regulatory role in the Smad signaling pathway as well. Here we investigate the performance of two homogeneous high-throughput screening assays capable of screening both modulators of receptor activation (e.g. agonists and antagonists) as well as intracellularly acting agents, such as inhibitors of upstream events. The assays were coupled to automated processes for increased throughput. Smad3 or Erk1/2 phosphorylation was measured following endogenous receptor activation in HeLa or HEK293 cell lines, respectively. The pharmacology of known inhibitors was also investigated.