FIG. 4.
The physical interaction between PU.1 and AML1 is localized to the DNA-binding domains. (A) AML1 (lanes 1 and 3 to 5) and AML1B (lanes 2 and 6 to 8) were transcribed and translated in vitro with [35S]methionine and incubated with glutathione-agarose (beads) (lanes 3 and 6), GST (lanes 4 and 7), or GST-PU.1 (lanes 5 and 8). Positions of molecular mass markers and the migration of the bound proteins are indicated to the left and right, respectively. (B) PU.1 was transcribed and translated in vitro with [35S]methionine and incubated with glutathione-agarose (beads) (lanes 1 and 6) or the following proteins immobilized on agarose: GST (lanes 2 and 7), GST-CBFβ (lanes 3 and 8), GST-runt (lanes 4 and 9), and GST-AML1B(213-395) (lanes 5 and 10). Ethidium bromide (EthBr; 50 μg/μl) was added to the reactions in lanes 6 to 10. Positions of molecular mass markers are shown to the left, and the migration of PU.1 is indicated to the right. (C) Full-length PU.1 (lanes 1 and 5 to 7), amino acids 161 to 272 of PU.1 containing the ets domain (lanes 2 and 8 to 10), and amino acids 1 to 163 of PU.1 (lanes 3 and 11 to 13) were transcribed and translated in vitro with [35S]methionine and incubated with glutathione-agarose (beads) (lanes 5, 8, and 11), immobilized GST (lanes 6, 9, and 12), or GST-runt (lanes 7, 10, and 13). Positions of the molecular mass markers, run in lane 4, are shown to the left, and the migration of the wild-type (wt) and mutant forms of PU.1 is indicated to the right.