Thermo Scientific™ LanthaScreen™ ERK2 A375 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 Erk 1/2
• Activated MEK phosphorylates Erk2 (Mapk1) proteins at a specific Thr-Tyr motif (Thr/Tyr 185/187)
• A number of constitutive active kinase mutants within the MAP kinase pathway have been implicated in oncogenesis
• One of these mutants is BRAF(V600E), which is the most predominant oncogenic BRAF mutant
• The human melanoma cell line A375 endogenously expresses BRAF(V600E), which leads to the constitutive activation of the MAP kinase pathway and phosphorylation of Erk2 in the absence of ligands
• LanthaScreenTM Erk2 A375 constitutively expresses GFP-Erk2 under control of a CMV promotor
• 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
• This cell line can be used to evaluate compound activity against BRAF(V600E)
• GFP-Erk2 lentivirus was transduced into A375 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.