ChIP assay for human separase promoter region. (A) U937T-AML1-ETO cells cultured in the presence or absence of tetracycline (Tet) for 24 hours were used in ChIP assay. The induction of AML1-ETO expression and a decrease of endogenous AML1 were detected by immunoblotting using anti-AML1 antibody. ChIP assay was performed with anti-AML1 antibody and bound DNA was examined by real-time quantitative PCR to show the binding of AML1-ETO to the upstream region of the separase gene. The relative binding activity was calculated by Ct value differences of ChIP DNA normalized with input DNA and that of cells without AML1-ETO was set to 1. The results are presented as the mean value of 3 independent experiments + standard deviation. (B) ChIP assay was performed with parental (P) and 3xFLAG-AML1-ETO9a (AE9a) stably transfected K562 cells using anti-FLAG M2 agarose. Immunoprecipitation of 3xFLAG-AML1-ETO9a was confirmed by immunoblotting with anti-FLAG antibody. The results of qPCR with ChIP DNA are shown as the mean value of 3 independent experiments + standard deviation. The relative binding activity was calculated by Ct value differences of ChIP DNA normalized with input DNA and that of parental cells was set to 1. Statistically significant P value compared with control (t test, P = .03) is indicated. (C) ChIP assay was performed with anti-FLAG M2 agarose using 293T cells transfected with wild-type (TC) phSeparase-luc or vectors with mutated AML1-binding sites (mC or Tm). pFLAG (C) or pFLAG-AML1-ETO (AE) vector was cotransfected. The expression (input) and immunoprecipitation (IP) of FLAG-AML1-ETO was confirmed by immunoblotting using anti-FLAG antibody (top). The relative binding of AML1-ETO to phSeparase-luc was examined by qPCR and it was calculated by Ct value differences of ChIP DNA normalized with input DNA. That of AML1-ETO–free sample (C) for each phSeparase-luc construct was set to 1 (bottom). The results are presented as the mean value of 3 independent experiments + standard deviation.