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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 LanthaScreenTM 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 LipofectamineTM 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.
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