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. 2010 Mar;1(3):210–224. doi: 10.1177/1947601910366860

Figure 3.

Figure 3.

HMGA1 binds a region upstream of the miR-181b gene. (A) Schematic representation of the genomic locus of miR-181b and of all putative conserved HMGA1b binding sites (indicated as A, B, C, and D) and analyzed in ChIP experiments. (B) ChIP of a miR-181b upstream genomic region. Semiquantitative PCR of the region flanking the putative HMGA1b binding sites (indicated as A in panel A) in the miR-181b upstream genomic region was performed to evaluate the amount of chromatin obtained by immunoprecipitation relative to the amount in the input samples. (C) Oligonucleotide pull-down assay. Proteins were extracted from MCF7 cells stably transformed with a pRcCMV/HMGA1b vector. The pull-down assay was performed as described in the Materials & Methods section. The samples were denatured and resolved on SDS-PAGE, and blots were probed with HMGA1-specific antibodies. (D) The same oligonucleotide pull-down experiment was performed with nonbiotinylated competitor oligonucleotides. We used a 10-fold excess of oligo 1 nonbiotinylated for nonspecific competition with respect to oligo 5-biotinylated (lane 4) and 10-fold excess of oligo 5-nonbiotinylated for specific competition with respect to oligo 5-biotinylated (lane 3).