Heterodimerization with PEBP2-β/CBF-β promotes phosphorylation of RUNX1 in a DNA binding-dependent manner. (A) PEBP2-β/CBF-β coexpression induces covalent modification of RUNX1 proteins in SDS-PAGE. HEK293 cells were transfected with plasmids for either RUNX1 (100 ng) alone or with PEBP2-β1 (300 ng). Twenty-four hours after transfection, cells were lysed and analyzed by Western blotting using an anti-RUNX1 or anti-PEBP2-β antibody. (B) Transfected HEK293 cell lysates were incubated in the presence or absence of calf intestine alkaline phosphatase (CIAP) followed by Western blotting using an anti-RUNX1 antibody. Phosphorylated RUNX1 proteins (P-RUNX1) and dephosphorylated/unphosphorylated RUNX1 (RUNX1) are indicated accordingly. (C) Ascertaining the phosphorylation status of endogenous RUNX1 in HL60, U937, and K562. Lysates from resting cells were treated with control or CIAP and analyzed by Western blotting using an anti-RUNX1 antibody. (D) Mapping of region in PEBP2-β responsible for the mediation of RUNX1 phosphorylation. HEK293 cells were transfected with RUNX1 (100 ng) and the indicated FLAG-PEBP2-β expression vectors (300 ng). The cells were harvested after 24 hours and analyzed by Western blotting to determine the phosphorylation status of RUNX1 by PAGE electrophoretic mobility. PEBP2-β1, -β2, and -β3 are naturally occurring isoforms of the PEBP2-β subunit. The β110 deletant is unable to dimerize with RUNX1. (E) Effects of RUNX1 point mutants found in AML patients or generated artificially. Wild-type and mutant RUNX1 proteins were coexpressed with PEBP2-β and processed as described in panel A. AML patient-derived mutants RUNX1^R174Q and RUNX1^K83E lack DNA binding ability, whereas an artificial mutant RUNX1^G108R lacks the ability to heterodimerize with PEBP2-β but is able to bind to DNA.