FIGURE 2.

Analysis of the responsive element to AML1b in the c-mpl promoter. A, EMSA was performed with the probe containing putative AML1-binding sequence in the human c-mpl promoter or known AML1-binding sequence (positive control). Nuclear extract was isolated from 293T cells transfected with the indicated genes and subjected to EMSA. In competition assays, a 1000-fold molar excess of unlabeled wild-type or mutant competitor oligonucleotide was added to the binding mixture. B, location of AML1-binding site and the primer set in the c-mpl promoter utilized for the ChIP assay are indicated. The nuclear extract was isolated from primary cultured murine hematopoietic cells and CMK cells, and the chromatin was sonicated. Then AML1-DNA-binding complexes were immunoprecipitated with the anti-AML1 Ab (N-20) or control goat IgG. The immunoprecipitated DNA was eluted and subjected to the PCR analyses. PCR products were electrophoresed on agarose gels and visualized with SYBR Green staining.