Comparison of the affinities for PEBP2β between the chimeric proteins and wild-type AML1. (A) COS-7 cells (1 × 10⁶) were transfected with the expression plasmid for HA-AML1, HA-AML1/ETO(MTG8), or HA-AML1/Evi-1. The amounts of the transfected expression plasmids were the same as described in the legend to Fig 6. The cells were lysed and incubated with GST (lanes 4 to 6) or GST-PEBP2β (lanes 7 to 9) linked to glutathione sepharose beads and subjected to Western blotting with anti-HA serum. Ten percent of the input was also run on the same gel (lanes 1 to 3). The positions of wild-type AML1 and the chimeric proteins are indicated by the arrowheads. Molecular weight standards (in kilodaltons) are shown. (B) COS-7 cells (1 × 10⁶) were transfected with the expression plasmid for PEBP2β together with the construct for HA-AML1 and that for HA-AML1/ETO(MTG8) or HA-AML1/Evi-1. The amounts of the transfected expression plasmids were the same as described in the legend to Fig 6. COS-7 cells transfected with only PEBP2β construct were also analyzed (mock). Cells were subjected to [³⁵S]methionine labeling and lysed. (a) Expressions of the HA-tagged chimeric proteins and wild-type AML1. Total cell lysates, including 50 μg of protein, were subjected to SDS-PAGE and Western blotting with anti-HA serum. Closed arrowheads show the position of HA-AML1. Open arrowheads show the positions of HA-AML1/ETO(MTG8) (lane 2) and HA-AML1/Evi-1 (lane 3). Molecular weight standards (in kilodaltons) are indicated. (b) Comparison of the amounts of the HA-tagged chimeric proteins to that of HA-AML1 immunoprecipitated with anti-HA monoclonal antibody. One milligram of each cell lysate was immunoprecipitated with anti-HA monoclonal antibody and subjected to SDS-PAGE and autoradiography. Closed arrowheads show the position of HA-AML1. Open arrowheads show the positions of HA-AML1/ETO(MTG8) (lane 2) and HA-AML1/Evi-1 (lane 3). The radioactivities of the bands of HA-AML1, HA-AML1/ETO(MTG8), and HA-AML1/Evi-1 were quantified by Fujix BAS 2000 (Fuji Film Corp, Kanagawa, Japan), and the ratios of the radioactivities of the HA-tagged chimeric proteins to those of HA-AML1 are indicated. Molecular weight standards (in kilodaltons) are shown. (c) Comparison of the amounts of the HA-tagged chimeric proteins to that of HA-AML1 immunoprecipitated with anti-PEBP2β serum. One milligram of each cell lysate was immunoprecipitated with rabbit anti-PEBP2β serum and subjected to SDS-PAGE and autoradiography. Closed arrowheads show the position of HA-AML1. Open arrowheads show the positions of HA-AML1/ETO(MTG8) (lane 2) and HA-AML1/Evi-1 (lane 3). The position of PEBP2β is marked by the arrow. The radioactivities of the bands of HA-AML1, HA-AML1/ETO(MTG8), and HA-AML1/Evi-1 were quantified by Fujix BAS 2000 (Fuji Film), and the ratios of the radioactivities of the HA-tagged chimeric proteins to those of HA-AML1 are indicated. Molecular weight standards (in kilodaltons) are shown.