Substitution of critical amino acids disrupts RUNX1/ETO tetramers into dimers without affecting its α-helical structure. (A) Size-exclusion chromatography of RUNX1/ETOtr (REtr) and the indicated mutant forms. Proteins were expressed in 293T cells and fractionated on a superose-6 column. Fractions were analyzed by Western blotting against the Flag epitope. (B) Signal intensity quantification of REtr, REtr-m5, and REtr-m7 elution profiles. (C) CD spectroscopy of the WT NHR2 and m5 mutant protein domains. (D) Ellipticity profile of NHR2 and NHR2-m5 at increasing temperature.