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Thermo Scientific™ LanthaScreen™ STAT5 (JAK2 V617F) U2OS Cell Line

Catalog No. K1588
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Description

LanthaScreen™ GFP Cellular Assays allow for the analysis of post-translational modifications for a number of target proteins in an live-cell context.

LanthaScreen™ GFP Cellular Assays allow for the analysis of post-translational modifications for a number of target proteins in an live-cell context. The JAK2/STAT5 signaling pathway plays an essential role in blood cell formation in response to cytokines such as GM-CSF, IL-3, and EPO. The recent discovery of an activating mutation in JAK2 (V617F) present in high percentage of myeloproliferative disease (MPD) patients suggests that this mutant JAK2 activity is a potential therapeutic target for certain forms of MPD.

The assay described in this summary makes use of a cell line engineered for expression of the constitutively-active mutant kinase JAK2 V617F. By co-expressing GFP-STAT5 in this background, the phosphorylation state of STAT5 (specifically residue Tyr 694/699) can be modulated with JAK2 V617F inhibitors and analyzed in cell lysates using an anti-STAT5 [pTyr 694/699] and LanthaScreen™ terbium-anti-mouse antibody pair. GFP-STAT5 Lentivirus was transduced into U2OS cells followed by selection with Blasticidin. The selected pool was then transfected with a GST-JAK2 V617F construct using Lipofectamine™ LTX, followed by selection with Geneticin.

This cell line is a clonal population isolated by flow cytometry using GFP fluorescence as sorting marker, and has been screened for the constitutive expression of GFP-STAT5 and GST-JAK2 V617F. Using a lytic TR-FRET immuno-assay, this cell line is validated for IC50 and Z' under optimized conditions using JAK Inhibitor I (Pyridone 6) as a small molecule inhibitor for JAK2 V617F-mediated GFP-STAT5 phosphorylation. This assay has also been tested for assay performance under variable experimental conditions, including cell plating density, stimulation time, DMSO tolerance and lysis/equilibration time.

TRUSTED_SUSTAINABILITY

Specifications

Alternate Agonists Constitutive
Antagonists JAK Inhibitor
Assay Entry Cell-based terbium phospho-specific activity (stable)
Cell State Dividing Cells
Content And Storage LanthaScreen™ STAT5 (JAK2 V617F) U2OS cells are shipped on dry ice. Store in liquid nitrogen immediately upon receipt, or thaw for immediate use.
Detection Method Fluorescence
Gene Symbol STAT5A
Optimal Response Constitutively Active
Primary Agonist Constitutive
Product Line LanthaScreen
Quantity 1 Vial
Readout TR-FRET
Reporter Gene GFP (EmGFP)
Target Entry STAT5
Cell Line U2OS
System Type LanthaScreen™
Druggable Target Signaling Pathway
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For Research Use Only. Not for use in diagnostic procedures.

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