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

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

Brand:  Thermo Scientific™ K1588

Product Code. 10318053

Description

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.

Specifications

• Alternate Agonists: Constitutive
• Assay Entry: Cell-based terbium phospho-specific activity (stable)
• 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.
• For Use With (Application): Kinases, Signaling Pathway
• Optimal Response: Constitutively Active
• Product Line: LanthaScreen™
• Readout: TR-FRET
• Target Entry: STAT5
• Cell Line: U2OS
• Druggable Target: Signaling Pathway

• Antagonists: JAK Inhibitor
• Cell State: Dividing Cells
• Detection Method: Fluorescence
• Gene Symbol: STAT5A
• Primary Agonist: Constitutive
• Quantity: 1 Vial
• Reporter Gene: GFP (EmGFP)
• Type: Dividing Cells
• System Type: LanthaScreen™

For Research Use Only. Not for use in diagnostic procedures.