Figure 4.
Figure 4. GABP binds to the FCAR promoter Ets-binding site. (A) EMSA of the FCAR promoter Ets-binding site using nuclear extracts. The labeled double-stranded oligonucleotide corresponding to the FCAR promoter sequence from -98 to -79 (wtETS-s) was incubated without (lane 1) and with 6 μg nuclear extracts from U937 (lanes 2-4), Jurkat (lanes 5-7), and HeLa cells (lanes 8-10) in the absence (lanes 1, 2, 5, and 8) or presence of 100-fold molar excess of unlabeled wild-type (wtETS-s; W) (lanes 3, 6, and 9) and mutant FCAR Ets oligonucleotide competitors (mutETS-s; M) (lanes 4, 7, and 10). Arrow indicates the location of HEL-NF1. Newly detected binding species (complexes I, II, III, and IV) are also indicated on left. Right panel shows long exposure of lanes 8 to 10 to show complex I. (B) EMSA of the FCAR promoter Ets-binding site using in vitro-translated GABP. The labeled double-stranded oligonucleotide corresponding to the sequence from -98 to -79 (wtETS-s) was incubated without (lane 1) and with 1 μL unprogrammed reticulocyte lysate (lane 2), 1 μL in vitro-translated GABPα (lane 3), 1 μL each of in vitro-translated GABPα and GABPβ (lanes 4-6), or 6 μg nuclear extracts from U937 (lane 7), Jurkat (lane 8), and HeLa cells (lane 9) in the absence (lanes 1-4, 7-9) or presence of 100-fold molar excess of unlabeled wild-type (wtETS-s; W) (lane 5) and mutant FCAR Ets oligonucleotide competitors (mutETS-s; M) (lane 6). Arrow indicates the location of HEL-NF1. Complexes I and III detected in nuclear extracts are also indicated on right. (C) Supershift EMSA of U937 nuclear extracts using the FCAR promoter Ets-binding site. The labeled double-stranded oligonucleotide corresponding to the sequence from -98 to -79 (wtETS-s) was incubated with 6 μg nuclear extracts from U937 cells in the presence of rabbit IgG (lane 1) or 1 μL antiserum against GABPβ (lane 2). Arrow indicates the location of HEL-NF1. (D) ChIP assay of HA-tagged GABPα binding in vivo. Crosslinked protein-DNA complexes from U937/HA-GAα cells were immunoprecipitated without antibody (lanes 2 and 7), with control rabbit IgG (lanes 3 and 8), and with antibodies against HA (lanes 4 and 9) and C/EBPα (lanes 5 and 10), and they were analyzed by PCR using primers corresponding to the FCAR promoter region from -143 to +8 (lanes 1-5) and a region within the FCAR EC1 exon (lanes 6-10). Positive control is chromatin prior to immunoprecipitation (lanes 1 and 6).

GABP binds to the FCAR promoter Ets-binding site. (A) EMSA of the FCAR promoter Ets-binding site using nuclear extracts. The labeled double-stranded oligonucleotide corresponding to the FCAR promoter sequence from -98 to -79 (wtETS-s) was incubated without (lane 1) and with 6 μg nuclear extracts from U937 (lanes 2-4), Jurkat (lanes 5-7), and HeLa cells (lanes 8-10) in the absence (lanes 1, 2, 5, and 8) or presence of 100-fold molar excess of unlabeled wild-type (wtETS-s; W) (lanes 3, 6, and 9) and mutant FCAR Ets oligonucleotide competitors (mutETS-s; M) (lanes 4, 7, and 10). Arrow indicates the location of HEL-NF1. Newly detected binding species (complexes I, II, III, and IV) are also indicated on left. Right panel shows long exposure of lanes 8 to 10 to show complex I. (B) EMSA of the FCAR promoter Ets-binding site using in vitro-translated GABP. The labeled double-stranded oligonucleotide corresponding to the sequence from -98 to -79 (wtETS-s) was incubated without (lane 1) and with 1 μL unprogrammed reticulocyte lysate (lane 2), 1 μL in vitro-translated GABPα (lane 3), 1 μL each of in vitro-translated GABPα and GABPβ (lanes 4-6), or 6 μg nuclear extracts from U937 (lane 7), Jurkat (lane 8), and HeLa cells (lane 9) in the absence (lanes 1-4, 7-9) or presence of 100-fold molar excess of unlabeled wild-type (wtETS-s; W) (lane 5) and mutant FCAR Ets oligonucleotide competitors (mutETS-s; M) (lane 6). Arrow indicates the location of HEL-NF1. Complexes I and III detected in nuclear extracts are also indicated on right. (C) Supershift EMSA of U937 nuclear extracts using the FCAR promoter Ets-binding site. The labeled double-stranded oligonucleotide corresponding to the sequence from -98 to -79 (wtETS-s) was incubated with 6 μg nuclear extracts from U937 cells in the presence of rabbit IgG (lane 1) or 1 μL antiserum against GABPβ (lane 2). Arrow indicates the location of HEL-NF1. (D) ChIP assay of HA-tagged GABPα binding in vivo. Crosslinked protein-DNA complexes from U937/HA-GAα cells were immunoprecipitated without antibody (lanes 2 and 7), with control rabbit IgG (lanes 3 and 8), and with antibodies against HA (lanes 4 and 9) and C/EBPα (lanes 5 and 10), and they were analyzed by PCR using primers corresponding to the FCAR promoter region from -143 to +8 (lanes 1-5) and a region within the FCAR EC1 exon (lanes 6-10). Positive control is chromatin prior to immunoprecipitation (lanes 1 and 6).

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