Constitutive DNA binding activity of STAT3 and STAT1 in leukemia cells expressing Asp⁸¹⁶ mutant c-Kit.

Parental MO7e cells and cells transduced with the wild-type c-kit cDNA (kit^WT) were cultured without SCF for 5 hours. The G50 clone (a cytokine-independent revertant from MO7e cells containing the D816H c-kit mutation), MO7e cells transduced with the mutant c-kit cDNAs (kit^DHand kit^DN) and the AML cells bearing the D816N mutant c-kit were cultured in the absence of SCF. These cells were then stimulated with rhSCF (40 ng/mL) for 5 minutes. Cytosolic extracts were prepared from the cells before and after SCF stimulation and subjected to EMSA (see “Materials and methods”). In the left panel, some samples were preincubated with a 50-fold excess of unlabeled (cold) probe, rabbit preimmune serum (PS), or defined STAT (St) antibodies. Protein-DNA complexes corresponding to the specific STAT dimers are indicated. A nonspecific band, which could not be completed by the addition of cold probe and was preferentially present in MO7e cells bearing the mutant c-kit, is also indicated by an arrow in both left and right panels.