Fig. 2.
Fig. 2. Functional PU.1, C/EBP, and c-Myb binding sites are present in the human MBN promoter. / (A) Recombinant human PU.1, C/EBPs, and c-Myb specifically bind functional sites in the MBN promoter. Five micrograms of whole-cell extract from COS-7 cells either untransfected (lanes 1, 5, 15) or transfected with PU.1 (lanes 2-4), C/EBPα (lanes 6-8), C/EBPβ (lanes 9-11), C/EBPδ (lanes 12-14), or c-Myb (lanes 16-18) were preincubated with 2 μg poly(dIdC) and a 400-fold molar excess of the wild-type (wt) or mutated (mut) competitors as indicated, before addition of the specific probe (PU.1, lanes 1-4; C/EBP, lanes 5-14; and c-Myb, lanes 15-18). Binding reaction mixtures were assayed by EMSA. The asterisks mark unspecific complexes. Supershift experiments were performed with 1 μL c-Myb antibody (Ab) and unrelated control Ab (lanes 19-22). Arrowhead indicates the specific supershifted complex. (B) Myeloid nuclear factors bind to the PU.1 and C/EBP sites. Nuclear extracts (1 μg) from exponentially growing PLB-985 cells were subjected to EMSA (lanes 1-7 and 11-19). DNA binding assays were performed with the PU.1 (lanes 1-10) or the C/EBP (lanes 11-24) probe in the presence of 5 μg and 0.2 μg poly(dIdC), respectively. Competition assays were carried out with a 40- and 200-fold molar excess of the wt or mut oligonucleotides. Arrows indicate the specific PU.1 or C/EBP complexes. Supershift experiments were performed with 1 μL anti-PU.1 (PU.1 Ab) (lanes 7-9), anti-C/EBPα, anti-C/EBPβ, anti-C/EBPδ (C/EBPα, C/EBPβ, C/EBPδ Abs) (lanes 17-19) and unrelated (control Ab) Abs (lanes 6, 8, 16, 20). Arrowheads indicate specific supershifted complexes. (C) PU.1, C/EBP, and Myb binding sites are all critical for MBN promoter activity in PLB-985 cells. Wt or indicated point mutation constructs (PU.1mut, C/EBPmut, and Mybmut) were transfected into cells. Transfection efficiencies were normalized by cotransfection of a CMV-luciferase vector. CAT activities relative to the wt (−658) are presented for PU.1mut, C/EBPmut, and Mybmut constructs. Error bars indicate SD from at least 3 different experiments. (D) CBP/p300 or related molecules are involved in MBN expression. PLB-985 cells were transfected with 20 μg pMBN-658 alone (−) or together with 2 and 10 μg of expression vector encoding E1A (13S) or the E1A 12S NTdl814 mutant. The average of 3 experiments are shown as relative CAT activity with SD as indicated. (E) Both c-Myb and C/EBPδ can cooperate to activate the MBN promoter. Five micrograms of pMBN-194 was transfected into COS-7 cells alone (−) or together with 1 μg of the CMV, CMV-Myb, MSV, MSV-C/EBPα, MSV-C/EBPβ, MSV-C/EBPδ, pECE, or pECE-PU.1 expression vectors or the indicated combinations of these vectors. CAT activities were assayed after 40 hours. The mean activations and standard errors observed in 3 determinations are shown.

Functional PU.1, C/EBP, and c-Myb binding sites are present in the human MBN promoter.

(A) Recombinant human PU.1, C/EBPs, and c-Myb specifically bind functional sites in the MBN promoter. Five micrograms of whole-cell extract from COS-7 cells either untransfected (lanes 1, 5, 15) or transfected with PU.1 (lanes 2-4), C/EBPα (lanes 6-8), C/EBPβ (lanes 9-11), C/EBPδ (lanes 12-14), or c-Myb (lanes 16-18) were preincubated with 2 μg poly(dIdC) and a 400-fold molar excess of the wild-type (wt) or mutated (mut) competitors as indicated, before addition of the specific probe (PU.1, lanes 1-4; C/EBP, lanes 5-14; and c-Myb, lanes 15-18). Binding reaction mixtures were assayed by EMSA. The asterisks mark unspecific complexes. Supershift experiments were performed with 1 μL c-Myb antibody (Ab) and unrelated control Ab (lanes 19-22). Arrowhead indicates the specific supershifted complex. (B) Myeloid nuclear factors bind to the PU.1 and C/EBP sites. Nuclear extracts (1 μg) from exponentially growing PLB-985 cells were subjected to EMSA (lanes 1-7 and 11-19). DNA binding assays were performed with the PU.1 (lanes 1-10) or the C/EBP (lanes 11-24) probe in the presence of 5 μg and 0.2 μg poly(dIdC), respectively. Competition assays were carried out with a 40- and 200-fold molar excess of the wt or mut oligonucleotides. Arrows indicate the specific PU.1 or C/EBP complexes. Supershift experiments were performed with 1 μL anti-PU.1 (PU.1 Ab) (lanes 7-9), anti-C/EBPα, anti-C/EBPβ, anti-C/EBPδ (C/EBPα, C/EBPβ, C/EBPδ Abs) (lanes 17-19) and unrelated (control Ab) Abs (lanes 6, 8, 16, 20). Arrowheads indicate specific supershifted complexes. (C) PU.1, C/EBP, and Myb binding sites are all critical for MBN promoter activity in PLB-985 cells. Wt or indicated point mutation constructs (PU.1mut, C/EBPmut, and Mybmut) were transfected into cells. Transfection efficiencies were normalized by cotransfection of a CMV-luciferase vector. CAT activities relative to the wt (−658) are presented for PU.1mut, C/EBPmut, and Mybmut constructs. Error bars indicate SD from at least 3 different experiments. (D) CBP/p300 or related molecules are involved in MBN expression. PLB-985 cells were transfected with 20 μg pMBN-658 alone (−) or together with 2 and 10 μg of expression vector encoding E1A (13S) or the E1A 12S NTdl814 mutant. The average of 3 experiments are shown as relative CAT activity with SD as indicated. (E) Both c-Myb and C/EBPδ can cooperate to activate the MBN promoter. Five micrograms of pMBN-194 was transfected into COS-7 cells alone (−) or together with 1 μg of the CMV, CMV-Myb, MSV, MSV-C/EBPα, MSV-C/EBPβ, MSV-C/EBPδ, pECE, or pECE-PU.1 expression vectors or the indicated combinations of these vectors. CAT activities were assayed after 40 hours. The mean activations and standard errors observed in 3 determinations are shown.

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