AML1/ETO physically interacts with AML1 on chromatin. (A) In vivo interaction between AML1/ETO and AML1 with co-IP assays in Kasumi-1 (left) and U937-A/E/9/14/18 (right) cells. The anti-AML1 (C19; Santa Cruz) and anti-AML1/ETO (fusion point; Diagenode) antibodies were used for IP. The anti-ETO (C20; Santa Cruz) and anti-AML1 (N20; Santa Cruz) antibodies were used for western blotting. Asterisks indicate nonspecific bands. The second antibody used in western blotting of AML1 immunoprecipitated with AML1/ETO in Kasumi-1 cells was horseradish peroxidase-conjugated anti-goat IgG TrueBlot (eBioscience), which could not detect the immunoglobulin heavy and light chains. The second antibody used in the remaining western blotting was horseradish peroxidase-conjugated anti-goat IgG (Santa Cruz). (B) Confocal immunofluorescence micrographs showing the distribution of AML1/ETO (green) and AML1 (red) in Kasumi-1 cells. Scale bars, 10 μm. (C) Validation for the coexistence of AML1/ETO and AML1 on chromatin through re-ChIP assays. Multiple overlapped regions were selected and validated for factor binding by a first round of ChIP followed by a second round with a different antibody or with IgG as control. The AML1 specific anti-AML1 (C19) antibody and AML1/ETO specific anti-ETO (C20) were used for re-ChIP assays. An AML1 unique region (*) and an AML1/ETO unique region (^#) were included as negative controls. Error bars represent the SD of triplicate measurements.