EMSA for comparison of DNA-binding activity. (A) Relative levels of AML1 (A), AML1-ETO (AE), and AML1-ETO9a (AE9a) protein expression in nuclear extracts are shown. Control (C) was prepared from 293T cells transfected with vector. show the position of AML1-, AML1-ETO (AE)–, and AML1-ETO9a (AE9a)–specific bands. (B) EMSA with human M-CSF receptor probe containing the TGTGGT consensus and nuclear extracts shown in panel A was performed. Anti-HA antibody was added to lanes 5 through 7 and supershifts were detected (◀). (C) EMSA with wild-type human separase (TC) probe and nuclear extracts shown in panel A was performed. Anti-HA antibody was added to lanes 5-8 and supershifts were detected (◀). (D) Schematic diagram of the human separase 5′ upstream region, exon 1, and exon 2. Two AML1 consensus sites are present with a space of 2 nucleotides (TGTGGTagTGCGGT) in the 5′-upstream region. Mutants of AML1 consensus sites used in this study are also listed. The −2 kb of 5′-upstream region (bold line) is fused with luciferase for promoter analysis in Figure 4. EMSA with wild-type (TC) and mutant (mC and Tm) human separase probes and nuclear extracts shown in panel A was performed.