DNA-binding affinity assay. (A) Expression level of AML1 (A) and AML1-ETO (AE) was examined by immunoblotting with anti-HA antibody in nuclear extracts. Control (C) was prepared from 293T cells transfected with vector. (B) DNA-binding affinity assay using wild-type and mutant separase probes with nuclear extracts shown in panel A was performed. The protein binding was detected by immunoblotting with anti-HA antibody. show the position of both AML1 (A) and AML1-ETO (AE). (C) DNA affinity purification assay using wild-type and mutant separase probes with nuclear extracts shown in panel A was performed. AML1 reaction (10 μL) and AML1-ETO reaction (5 μL) were loaded. (D) DNA affinity purification assay with Kasumi-1 cells was performed. Input lane shows the relative expression of endogenous AML1 and AML1-ETO examined by immunoblotting with anti-AML1 antibody in nuclear extracts. The protein-binding activity of wild-type (TC) or mutant (mm) separase probes was examined by immunoblotting with anti-AML1 antibody and shown in IP lanes. (E) DNA affinity purification assay with Kasumi-1 cells using wild-type or mutant SPARC and PU.1 probes was performed. Input lane shows the relative expression of endogenous AML1 and AML1-ETO examined by immunoblotting with anti-AML1 antibody in nuclear extracts. The protein-binding activity of wild-type (AC for SPARC and CT for PU.1) or mutant (mm) probes was examined by immunoblotting with anti-AML1 antibody and shown in IP lanes. (F) Expression level of full-length (AE) and NHR2-deleted (AEΔ) AML1-ETO in nuclear extracts was examined by immunoblotting with anti-HA antibody (input, lanes 1 and 2). The results of DNA affinity purification assay using wild-type (TC) or mutant (mC) separase probes with full-length (lane 3) and NHR2-deleted (lanes 4-6) AML1-ETO are shown. show the position of both full-length (AE) and NHR2-deleted (AEΔ) AML1-ETO.