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. 1998 Jul;18(7):3915–3925. doi: 10.1128/mcb.18.7.3915

FIG. 5.

FIG. 5

FIG. 5

FIG. 5

FIG. 5

AML1B and PU.1 synergize to transactivate the M-CSF receptor promoter. (A) Synergistic transactivation of the M-CSF receptor promoter by PU.1 and AML1B requires intact binding sites for both factors. Transient transfections were performed in HeLa cells with 5 μg of reporter plasmid and 1 μg of expression plasmid containing CBFβ (pCMV5-CBFβ), 1 μg of each expression plasmid for PU.1 (PU.1pECE) or AML1B (pCMV5-AML1B), or empty vector. The results represent the mean activation of the promoter ± standard error of five experiments. In this set of experiments, the activity of pM-CSF-R-luc was three times higher than that of the empty vector, pXP2. (B) Mutations in the N terminus of PU.1 abrogate synergy with AML1B. The effect of wild-type (wt) PU.1 on pM-CSF-R-luc was compared to effects of mutations of PU.1 in the N terminus, in the presence or absence of AML1B. The names of the mutants represent the deleted amino acids. The results are normalized to the level of expression of pM-CSF-R-luc and represent the mean ± standard error of three experiments. Fold synergy is calculated by dividing the activation in the presence of both factors by the sum of the activation by each factor individually. (C) The C terminus of AML1B is required for synergy with PU.1. Transient transfections were performed with PU.1, CBFβ, and either AML1, AML1B, or mutants of AML1B in order to identify the region of AML1 required for functional interaction with PU.1. Mutants AML1B(1-289), -(1-317), and -(1-381) are carboxy-terminal truncations of AML1B; the numbers represent the amino acids which are encoded. The results are normalized to the level of expression of pM-CSF-R-luc and represent the mean ± standard error of three experiments. (D) COS-7 cells were either mock transfected (lane 1) or transfected with Lipofectamine Plus, 2 μg of expression plasmid encoding AML1B (lane 2), or AML1B truncated at amino acid 268 (1-268, lane 3), 289 (1-289, lane 4), 317 (1-317, lane 5), or 381 (1-381, lane 6) or with AML1 (lane 7). After 20 h, the cells were labeled with [35S]methionine and [35S]cysteine and immunoprecipitated, and the AML1 proteins were resolved by SDS-PAGE. Bands corresponding to the expected molecular masses are indicated with asterisks, and the migration of the molecular mass markers is indicated to the right.