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. Author manuscript; available in PMC: 2012 Mar 19.
Published in final edited form as: J Mol Endocrinol. 2011 Nov 21;47(3):299–313. doi: 10.1530/JME-11-0060

Figure 7.

Figure 7

Several proteins specifically bind to the −121/−83 bp region of the HAMP promoter. EMSA was used to assess the binding of nuclear protein extracts (7·5 μg) prepared from Huh7 cells to a double-stranded DIG-labeled oligonucleotide corresponding to the wild-type (WT) promoter region −121/−83 bp of the HAMP gene (A; the underlined sequence indicates the GATA-RE). Nuclear protein extracts from Huh7 cells formed specific complexes (I–III) with the probe as indicated to the left of the image (B). Protein binding was challenged by increasing molar concentrations (125× and 250×; black triangles) of unlabeled oligonucleotides corresponding to the WT probe and the mutant (MT), the sequences of the WT and mutant used are shown in Table 1. To evaluate binding of GATA proteins to the HAMP promoter (C) nuclear protein extracts 7·5 μg) were prepared from Huh7 cells 40 h following transfection with 24 μg of either an expression vector encoding the full-length WT GATA protein (GATA-4 (C, lanes 4 and 5) or GATA-6 (C, lanes 8 and 9)) or an empty plasmid vector (pcDNA3 (C, lanes 2, 3, 6, and 7)). Specific binding to a double-stranded DIG-labeled oligonucleotide corresponding to the WT promoter region −121/−83 bp of the HAMP gene (A) of nuclear extracts from Huh7 cells overexpressing either GATA-4 (C, lanes 4 and 5) or GATA-6 (C, lanes 8 and 9) protein was evaluated in the presence of either an antibody directed toward the overexpressed GATA protein (anti-GATA-4 (C, lane 5) or anti-GATA-6 (C, lane 9)) or normal goat serum as a control (C, lanes 4 and 8). Overexpressed GATA protein (4 or 6) incubated with the probe in presence normal goat serum showed a bigger complex II (C, lanes 4 and 6) than control nuclear extracts collected from Huh7 cells transfected with an empty vector [pCDNA3] that were incubated with the probe in presence of normal goat serum (C, lanes 2 and 6). Complex II was abrogated by incubation of the probe with nuclear extracts containing the overexpressed GATA protein along with an antibody directed against the corresponding overexpressed protein (goat anti-human GATA-4 polyclonal antibody or goat anti-human GATA-6 polyclonal antibody for GATA proteins 4 or 6, respectively), as shown in C, lane 5 (in the case of GATA-4) or lane 9 (in the case of GATA-6).