Thermo Scientific™ LanthaScreen™ ERK2 U2OS Cell Line

Description

• 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)
• LanthaScreen™ 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.