FIGURE 2.

Mutation of RUNX1 serines 48, 303, and 424 to aspartic acid to mimic cdk phosphorylation reduces HDAC1 and HDAC3 interaction in cell extracts. A, 293T cells were co-transfected with Myc-tagged wild-type RUNX1 (mycRUNX-WT), RUNX1(tripleA) (mycRUNX-3A), or RUNX1(tripleD) (mycRUNX1–3D) in the absence or presence of FLAG-tagged HDAC1 (FLAG-HDAC1), each expressed from the CMV promoter. Two days later, cell extracts were subjected to immunoprecipitation (IP) with rabbit anti-Myc antiserum or normal rabbit Ig followed by Western blotting (WB) using mouse anti-FLAG or anti-Myc antibodies (left panels). Equal amounts of cell extracts were also analyzed by Western blotting prior to immunoprecipitation to assess input protein expression (right panels). Co-immunoprecipitated FLAG-HDAC1 band intensities were quantified, as shown below each lane of upper left panel. B, similar analysis was conducted substituting FLAG-tagged HDAC3 (FLAG-HDAC3) for FLAG-HDAC1.