Constitutive STAT3 activity in AML blasts.

(A) The top panel shows EMSA, demonstrating the SIE-binding activity of STAT3, and the lower 2 panels show Western blotting. The middle panel shows hybridization with antibodies against phosphorylated STAT3, and the lower panel shows hybridization with anti-STAT3 antibodies to demonstrate equal loading. The positions of STAT3α and STAT3β are indicated. Lanes 1, 2, 3, 5, and 6 show patient samples with constitutive STAT3 activity. Lane 4 represents a patient sample without constitutive STAT3 activity. Note that the bands in lanes 1, 2, 3, and 6 demonstrate faster migration—that is, STAT3α—whereas the band in lane 5 shows slower migration—that is, STAT3β. (B) Supershift analysis of the SIE band complex formed with C-terminally directed anti-STAT3 antibodies in cells from a patient expressing predominantly STAT3β and MO7E cells expressing predominantly STAT3α. There is no supershift in the patient sample, whereas STAT3α is supershifted in MO7E cells. (C) Western blot analysis of STAT3 hybridized with antibodies directed against the N-terminal (top panel) and the C-terminal (lower panel) domains. When using antibodies directed against the C-terminal domain of STAT3, STAT3β was not detected. To verify that no change occurred after cytokine exposure, samples were exposed to human G-CSF (10 ng/mL) for 10 minutes as previously described.13