Jak/Stat signaling pathways play essential roles in 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 TF1 Cell Line contains a beta - lactamase reporter gene under control of the irf - 1 response element stably integrated into TF1 cells. TF1 cells are a human erythroleukemia cell line that is growth - dependent on GM - CSF and have an intact GM - CSF - JAK2 - STAT5 pathway. This cell line is validated for EC50 and Z’ - factor using GM - CSF. This cell line has also been tested under variable experimental conditions, including DMSO concentration, cell number, stimulation time, and substrate loading time. Responsiveness to EPO and Jak Inhibitor 1 was also tested (Figures 2 and 3). CellSensor® irf1 - bla TF1 cells were stimulated in triplicate with GM - CSF over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with agonist and 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 were plotted for each replicate against the indicated concentrations of GM - CSF. CellSensor® irf1 - bla TF1 cells were stimulated with Epo over the indicated concentration range in a 384 - well format. Cells were incubated for 5 hrs with agonist and 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 Response Ratios were plotted against the indicated concentrations of Epo. CellSensor® irf1 - bla TF1 cells were treated with Jak Inhibitor 1 over the indicated concentration range in a 384 - well format. Cells were then stimulated with GM - CSF or EPO for 5 hrs 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. These values were converted to percent inhibition relative to a set of controls (unstimulated cells and EC80 of GM - CSF or EPO treated cells) and plotted against the indicated concentrations of Jak Inhibitor 1. Academic and non-profit customers, please inquire for special pricing.
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