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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. In hematopoeitic cells, 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. In this pathway, binding of these cytokines to their respective cell surface receptors results in the activation of JAK2, which in turn phospho-activates STAT5 proteins at specific tyrosine residues (Tyr-694⁄699). LanthaScreenTM-STAT5 TF-1 is a human hematopoeitic cell line which constitutively expresses GFP-STAT5 fusion proteins. The JAK2⁄STAT5 signaling pathway is known to be functionally intact in this cell line, therefore the GFP-STAT5 fusion protein serves as a substrate for the inducible phosphorylation by JAK2. Using this cell line, a homogenous immuno-assay has been developed in which the phosphorylation state of GFP-STAT5 is detected in cell lysates using a LanthaScreenTM Terbium-anti-mouse and anti-phospho STAT5 [pTyr694⁄699] antibody pair, in a time-resolved FRET (TR-FRET) readout. GFP-STAT5 Lentivirus was transduced into TF-1 cells followed by selection with Blasticidine. 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 fusion protein. Using a lytic TR-FRET immuno-assay, this cell line is validated for EC50 and Z' under optimized conditions using GM-CSF as an agonist for JAK2 -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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