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The Map Kinase (MapK) signal transduction cascade is activated by growth factors such as EGF, PDGF, and HGF. Binding of these factors to their respective cell surface receptors results in the initiation of receptor tyrosine kinase activity, which leads to the sequential phospho-activation of downstream kinases such as Ras, Raf, MEK, and Erk1⁄2. Activated MEK phosphorylates Erk2 (Mapk1) proteins at a specific Thr-Tyr motif (Thr⁄Tyr 185⁄187). LanthaScreenTM Erk2 U2OS is a human cell line which constitutively expresses GFP-Erk2. The MapK signaling pathway is functionally intact in this cell line, therefore the GFP-Erk2 fusion protein serves as a substrate for the growth-factor-inducible phosphorylation by MEK. Using this cell line, a homogenous immuno-assay has been developed in which the phosphorylation state of GFP-Erk2 is detected in cell lysates using a terbium-labeled anti-pTpY-185⁄187-Erk2 antibody, in a time-resolved FRET (TR-FRET) readout. GFP-Erk2 lentivirus was transduced into U2OS cells, followed by selection with Blasticidin. This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as sorting marker. Using the lytic TR-FRET immuno-assay, this cell line is validated for EC50 and Z’ under optimized conditions using EGF as a ligand for GFP-Erk2 phosphorylation. This assay has also been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance and cell lysis⁄equilibration time. Additional information using alternate stimuli and small molecule inhibitor is also provided.
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