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Flow cytometry was performed with Alexa Fluor 647 mouse anti-pSTAT5Y694 (clone 47, BD Biosciences). MOLM-13 cells were cultured in CM for 4 hours prior to administration of quizartinib and candidate inhibitors. Inhibitors were identified by literature review of kinases implicated in STAT5 phosphorylation in AML. Cells were harvested following 4 hours of culture with inhibitor, fixed (BD Phosflow Fix Buffer I) and permeabilized (BD Phosflow Perm Buffer III). Cells were analyzed for fluorescence intensity on a BD LSRFortessa cell analyzer.
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