Thermo Scientific™ LanthaScreen™ STAT3 GripTite™ Cell Line

Description

• The JAK/STAT3 signaling pathway is known to be activated by cytokines such as IL-6
• In this pathway, binding of IL-6 to its cell-surface receptors results in the activation of JAKs, which in turn phosphoactivate STAT3 proteins at a specific tyrosine residue (Y705)
• LanthaScreen™ STAT3 GripTite™ is a human cell line that constitutively expresses a GFP-STAT3 fusion protein
• The JAK/STAT signaling pathway is known to be functionally intact in this cell line; therefore, the GFP-STAT3 fusion protein serves as a substrate for IL-6-inducible phosphorylation
• Using this cell line, a lytic immunoassay has been developed in which the phosphorylation state of GFP-STAT3 is detected in cell lysates using a terbium-labeled anti-pY705-STAT3 antibody in a time-resolved FRET (TR-FRET) readout
• The GFP-STAT3 DNA expression construct was transfected into the GripTite™ HEK 293 cell line using Lipofectamine™ 2000 transfection reagent, and the transfected cells were selected with blasticidin
• This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as a sorting marker
• The assay utilizing this cell line is validated for EC50 and Z'-factors under optimized conditions using IL-6 as a ligand for JAK-mediated GFP-STAT3 phosphorylation
• This assay has also been tested under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance, and assay equilibration time
• An alternate 'mix-and-read' assay format is also described.