Experimental evidence shows that AML1/ETO and AML1 bind to adjacent short and long motifs. (A) Direct comparison of the kinetics of AML1 and AML1/ETO binding on short and long motifs using biolayer interferometry. (Left upper) Sequence of biotin-labeled probes used in the assays. S1 and S2 represent the short AML1 motif-containing probes, whereas L1 and L2 represent the long AML1 motif-containing probes. (Left lower) Equilibrium dissociation constants (K_D). (Right) Association and the disassociation curves of each experiment group. The protein concentrations of AML1/ETO and AML1 used were 0 (light green), 14.8 (dark green), 44.6 (red), and 134 nM (blue). (B) DNA pull-down assays for AML1/ETO and AML1 with the long and short motif-containing probes. The probes used in DNA pull-down assays were the same as those used in biolayer interferometry experiments. The equal amount of AML1/ETO and AML1 was used in DNA pull-down assays (left). The protein binding was detected by western blotting with anti-AML1 (N20; Santa Cruz) antibody. Data using additional 2 short and 2 long AML1 motif-containing probes can be found in supplemental Figure 4A. (C) ChIP-qPCR analyses of AML1/ETO and AML1 recruitment on the short and long motif-containing regions in Kasumi-1 cells. Data using additional 2 short and 2 long AML1 motif-containing regions can be found in supplemental Figure 4B. Error bars represent the standard deviation (SD) of triplicate measurements.