Jak/Stat signaling pathways play essential roles in the cellular responses to distinct cytokines. One of the Jak/Stat pathways, Jak2/Stat5, mediates cell proliferation in response to Interleukin - 3 (IL - 3), prolactin, erythropoietin (Epo), and granulocyte - macrophage colony stimulating factor (GM - CSF). JAK2 gene knockout causes embryonic lethality due to defective erythropoiesis, suggesting that the Jak2/Stat5 pathway plays an important role in red blood cell formation. The recent discovery of an activating mutation in JAK2 (V617F) present in a high percentage of myeloproliferative disease (MPD) patients suggests that the Jak2/Stat5 pathway may be a potential therapeutic target for certain forms of MPD. The activated Stat5 transcription factor recognizes and binds to a specific palindromic DNA sequence found in the promoter region of β - casein, interferon regulatory factor - 1 (irf - 1) and a number of other genes. The CellSensor® irf1 - bla CTLL2 Cell Line contains a beta - lactamase reporter gene under control of the irf - 1 response element stably integrated into CTLL2 cells. CTLL2 cells are a clone of cytotoxic T cells derived from a C57BL/6 mouse, and are cell - growth dependent on mouse Interleukin - 2 (mIL - 2). This cell line is validated for EC50 and Z' - factor under optimized conditions using mIL - 2. This cell line has also been tested under variable assay conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Responsiveness to pathway inhibitors was also tested. CellSensor® irf1 - bla CTLL2 cells were stimulated with mIL - 2 in triplicate over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with agonist in 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2.5 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader. The 460/530 ratios for each replicate plotted against the indicated concentrations of mIL - 2. CellSensor® irf1 - bla CTLL2 cells were treated with Jak Inhibitor 1 over the indicated concentration range in a 384 - well format for 30 min. Cells were then incubated for 5 hrs with mIL - 2 agonist (1 ng/ml) in 0.5% DMSO and then combined with LiveBLAzer™ - FRET B/G Substrate (CCF4 - AM) for 2 hrs. Fluorescence emission values at 460 nm and 530 nm were obtained using a standard fluorescence plate reader, converted to percent inhibition relative to a set of controls (unstimulated cells and EC80 stimulated cells), and plotted against the indicated concentrations of Jak Inhibitor 1. Academic and non-profit customers, please inquire for special pricing.