Constitutive activation of STAT5 by Flt3-ITD.
(A) Detection of STAT5 activation by immunoblot. Total cell lysates were separated by SDS-PAGE and immunoblotted with anti-pSTAT5, which reacts with STAT5 phosphorylated on tyrosine 694, or antitotal STAT5 as indicated. (B) Electrophoretic mobility shift assay of STAT5. Nuclear extracts were incubated with a double-stranded 32P-labeled oligonucleotide containing the consensus STAT5 binding sequence. Results from 2 constructs are shown: ITD1 (lanes 1-5) and Flt3-WT (lanes 6-10). The stimulation conditions of the cells before lysis are indicated: no growth factors added (N, lanes 1 and 6), 10 ng/mL IL-3 for 10 minutes (IL-3, lanes 2, 3 and 7, 8), and 100 ng/mL FL (FL, lanes 4, 5 and 9, 10). To verify the presence of STAT5 in the shifted oligonucleotide/protein complex, a supershift assay was performed with an anti-STAT5a/b antibody as indicated (lanes 3, 5 and 8, 10). Note that no shift or supershift is visible in unstimulated and FL-stimulated wild-type Flt3 cells, whereas shifted complexes containing STAT5 can be seen in ITD1 cells.
