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. 2011 Feb 9;39(11):4640–4652. doi: 10.1093/nar/gkr023

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© The Author(s) 2011. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Analysis of the association of C/EBPβ and p300 with the mbr and the SBS1 sequence with ChIP and EMSA. (A) ChIP assay showed C/EBPβ (lane 3) and p300 (lane 4), but not CBP (lane 5), associated with the mbr in vivo. The PCR products amplified by specific primers for the mbr were visualized on 1.5% agarose gel. (B) ChIP analysis showed that C/EBPβ (lane 3) and p300 (lane 4), but not CBP (lane 5), associated with SBS1 in vivo. The DNA immunoprecipitated with different antibodies were amplified by specific primers for the SBS1. (C) EMSA demonstrated that C/EBPβ, p300 and SATB1 formed a protein complex at 42 mbr. Lane 1: the negative control without cell lysate. Lane 2: protein complexes formed at 42 mbr. Lane 3: 100-fold molar excess of the cold 42 mbr probe successfully competed with hot 42 mbr probe. Lane 4: 100-fold molar excess of the cold SP1 consensus sequence (the non-specific probe) failed to compete with the labeled 42 mbr that was bound by the protein complexes. Lane 5, 6, 7: the supershift experiment with 2, 6 and 10 μg anti-C/EBPβ antibody respectively. Lane 8, 9, 10: the supershift experiment with 2, 6 and 10 μg anti-p300 antibody respectively. Lane 11, 12, 13: the supershift experiment with 2, 6 and 10 μg anti-SATB1 antibody respectively. The arrow indicated the bands supershifted from the same DNA–protein complex with addition of three different antibodies. (D) EMSA demonstrated that SATB1, but not C/EBPβ or p300 binding to the SBS1 in vitro. Lane 1: the negative control without cell lysate. Lane 2: protein complexes formed at SBS1. Lane 3: the bands were successfully competed with 100-folds molar excess of the cold SBS1 probe. Lane 4: 100-folds molar excess of cold SP1 consensus sequence failed to compete with the SBS1 to be bound by the protein complexes. Lane 5, 6, 7: the supershift experiment with 2, 6 and 10 μg anti-SATB1 antibody respectively. Lane 8, 9, 10: the supershift experiment with 2, 6 and 10 μg anti-p300 antibody respectively. Lane 11, 12, 13: the supershift experiment with 2, 6 and 10 μg anti-C/EBPβ antibody respectively. As indicated by the arrow, DNA–protein formation was inhibited and supershifted by addition of anti-SATB1 antibody, while no blocked or supershifted band was observed with addition of anti-C/EBPβ or anti-p300 antibodies.