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. 2013 Apr 12;27(12):2301–2310. doi: 10.1038/leu.2013.83

Figure 1.

Figure 1

FLT3-ITD induces STAT5 phosphorylation in HEK293 cells. (a) Strategy for the luciferase-based screening assay. FLT3-ITD-mediated STAT5 activation via phosphorylation (P) drives the expression of the firefly luciferase reporter system. The gray box indicates the internal tandem duplication (ITD) in the juxtamembrane domain of FLT3. (b) HEK293 cells stably expressing FLT3-ITD (ITD), FLT3 WT (WT), or no FLT3 (0) were analyzed with FLT3 and STAT5 phospho-specific antibodies (p). Blots were reprobed for total protein (t). The star indicates an unspecific, cross-reacting band. (c) HEK293 cells stably expressing FLT3-ITD were transfected with esiRNA-targeting FLT3 or GFP (negative control). Downregulation of FLT3 was analyzed by immunoblotting with an anti-FLT3 antibody. Activation of STAT5 was analyzed using an anti-P-STAT5 (p) antibody in comparison to an antibody detecting total STAT5 protein (t). (d) Luciferase activity of HEK293 FLT3 wild type or HEK293 FLT3-ITD cells is shown for cells transfected with the STAT5 reporter plasmid pLHRE-firefly-luciferase encoding firefly luciferase (left columns) and the control plasmid pRL-SV40-Renilla-luciferase (right columns), or non-transfected cells. Cells were transfected with identical amount of plasmid DNA. Luciferase assays were performed 72 hours post transfection.