(A) Expressions of the HA-tagged wild-type AML1 and chimeric proteins in COS-7 cells. A total of 3 × 104COS-7 cells were transfected with the expression plasmid for PEBP2β (0.4 μg) together with that for each HA-tagged protein. To obtain the same expression level of HA-AML1, HA-AML1/ETO(MTG8), and HA-AML1/Evi-1, based on several preparative experiments, we determined the amounts of the transfected plasmids as follows; HA-AML1 (0.5 μg), HA-AML1/ETO(MTG8) (1.2 μg), and HA-AML1/Evi-1 (4.0 μg). Cell lysates were fractionated into cytoplasmic (lanes C) and nuclear (lanes N) fractions and analyzed by immunoblotting using anti-HA serum. Wild-type AML1 and the chimeric proteins were expressed at the anticipated sizes, as marked by the arrowheads. The complete transfer of these three proteins was verified by confirming that all molecular weight standards were equally stained on the membrane and barely detectable on the gel by Coomassie staining after blotting (data not shown). Cell lysates of COS-7 cells transfected with only PEBP2β construct were also analyzed (mock). Molecular weight standards (in kilodaltons) are indicated. (B) (see page 1690) Double fluorescence labeling of COS-7 cells transfected with the constructs of each HA-tagged protein and PEBP2β. The amounts of the transfected expression plasmids were the same as indicated in (A). (a, b, c, and d) The HA-tagged proteins were detected with anti-AML1 serum. (e, f, g, and h) PEBP2β was detected with hamster anti-PEBP2β serum. Original magnification × 600. (C) The ability of each HA-tagged protein to accumulate PEBP2β in the nucleus. COS-7 cells were transfected with the expression plasmids as shown in (A). Two hundred cells expressing both each HA-tagged protein and PEBP2β were counted (see text). Bars show the percentages of the cells showing stronger fluorescence detected by anti-PEBP2β serum in the nucleus as compared with the fluorescence in the cytoplasm (obtained in three independent experiments). Error bars indicate one standard deviation. (D) The staining pattern of the cells with anti-PEBP2β serum overexpressing HA-AML1 (upper panel), HA-AML1/ETO(MTG8) (middle panel), or HA-AML1/Evi-1 (lower panel) in lower magnification (× 100).
