Identification of the constitutively activated DNA-binding factor in Dami/HEL and Meg-01 cells and the cytokine-induced DNA-binding factor in Mo7e cells by gel electrophoretic mobility supershift assays. Nuclear extracts prepared from Dami/HEL cells without cytokine exposure were incubated with [³²P]-labeled IRF-1 GAS plus anti-STAT3 or anti-STAT5 antibodies (lanes 1 and 2). Nuclear extracts from Meg-01 cells without cytokine exposure were incubated with the [³²P]-labeled probe plus anti-STAT3 or anti-STAT5 antibodies (lanes 3 and 4). Nuclear extracts from Mo7e cells without cytokine exposure (lane 5) or treated with 400 ng/mL TPO (lanes 6 to 8), were incubated with [³²P]-labeled IRF-1 GAS probe (lanes 5 and 6), or with [³²P]-labeled probe plus anti-STAT3 or anti-STAT5 antibodies (lanes 7 and 8). Nuclear extracts, from SK-BR–3 cells incubated without cytokine exposure (lane 9) or with 20 ng/mL EGF (lanes 10 to 14), were incubated with [³²P]-labeled IRF-1 GAS probe alone (lanes 9, 10, and 13), or with [³²P]-labeled probe plus anti-STAT1, anti-STAT3, or anti-STAT5 antibodies (lanes 11, 12, and 14). The EGF-treated SK-BR–3 cells, which were reported by our group to have activated STAT1, STAT3, and STAT5,36 were used to show the antibody-induced supershift of STAT1, STAT3, and STAT5. The autoradiograph shows the STAT-like DBF, NS, and free IRF-1 GAS probe (P). The arrowheads indicate the supershifted complexes. These results are representative of three separate experiments.