Thermo Scientific™ LanthaScreen™ STAT1 U2OS Cell Line

Description

• The JAK/STAT1 signaling pathway is known to be activated by type I/II interferons such as interferon-gamma and interferon-alpha
• In this pathway, binding of these cytokines to their respective cell-surface receptors results in the activation of JAKs, which in turn phosphoactivate STAT1 proteins at a specific tyrosine residue (Y701)
• LanthaScreen™ STAT1 U2OS is a human cell line that constitutively expresses a GFPSTAT1 fusion protein
• The JAK/STAT1 signaling pathway is known to be functionally intact in this cell line; therefore, the GFP-STAT1 fusion protein serves as a substrate for the IFN-gamma-inducible phosphorylation by JAKs
• Using this cell line, a homogeneous immunoassay has been developed in which the phosphorylation state of GFP-STAT1 is detected in cell lysates using a terbium-labeled anti-pY701-STAT1 antibody in a time-resolved FRET (TR-FRET) readout
• The GFP-STAT1 DNA expression construct was transfected into U2OS cells using Lipofectamine™ LTX Reagent, followed by selection with blasticidin
• This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as a sorting marker
• Using the lytic TR-FRET immunoassay, this cell line is validated for EC50 and Zfi-factors under optimized conditions using IFN-gamma as a ligand for JAK-mediated GFP-STAT1 phosphorylation
• This assay has also been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance, and assay development time
• Additional information using alternate stimuli and alternate assay protocols is available.