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-cell자세히 알아보기

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.

사양

대체 작용제Constitutive

길항제JAK Inhibitor

어세이 입력Cell-based terbium phospho-specific activity (stable)

셀 상태Dividing Cells

검출 방법Fluorescence

유전자 기호STAT5A

최적 반응Constitutively Active

일차 작용제Constitutive

제품라인LanthaScreen™

수량1 Vial

판독TR-FRET

리포터 유전자GFP (EmGFP)

타겟 입력STAT5

세포주U2OS

System TypeLanthaScreen™

Druggable TargetSignaling Pathway

Unit SizeEach