Skip to main content
. 2011 Apr 6;286(21):19047–19064. doi: 10.1074/jbc.M110.213983

FIGURE 8.

FIGURE 8.

Cdx4 bound to the HOXA10 promoter. A, Cdx4 bound to the HOXA10 promoter by a DAPA. The DAPA was performed using nuclear proteins from U937 cells and a biotin-labeled, double-stranded, synthetic oligonucleotide probe representing the −129 to −136 bp sequence of HOXA10 (or irrelevant control oligonucleotide). Proteins that co-purified with the oligonucleotide probes were separated by SDS-PAGE, and Western blots were probed with antibody (Ab) to HoxA10. Non-precipitated (input) nuclear protein was a control in this experiment. B, Cdx4 bound to the HOXA10 promoter by EMSA. EMSA was performed using nuclear proteins from U937 cells and a radiolabeled, double-stranded, synthetic oligonucleotide probe representing the −129 to −136 bp sequence of HOXA10. Binding assays were incubated with an irrelevant control antibody or Cdx4 antibody or with double-stranded, unlabeled oligonucleotide competitors representing homologous −129 to −136 bp HOXA10 sequence, −129 to −136 bp HOXA10 sequence with mutation of the Cdx4-binding consensus, the −139 to −150 bp HoxA10-binding CDX4 sequence, a non-HoxA10-binding mutant form of the −139 to −150 bp CDX4 sequence, or an irrelevant oligonucleotide from the CYBB gene, as indicated. The arrow represents the Cdx4 DNA-bound complex. The first lane represents free probe in a reaction with no nuclear proteins or oligonucleotide competitors. C, Cdx4 bound a cis element in the HOXA10 promoter in vivo. Chromatin immunoprecipitation was performed with U937 cells and antibodies to Cdx4 or HoxA10 (or irrelevant control antibody). Co-precipitating chromatin was amplified by real-time PCR with primers that flank the −45 to −160 bp HOXA10 promoter sequence (the functional cis element), the −400 to −500 bp HOXA10 promoter sequence (an irrelevant region of HOXA10), the −630 to −750 bp HOXA10 promoter sequence (another irrelevant region of HOXA10), or an irrelevant, non-Cdx4-binding cis element from the PTPN13 gene. ChIP results were normalized to PCR with non-precipitated, input chromatin to control results between experiments. *, statistically significant difference in Cdx4 binding versus nonspecific binding to the HOXA10 probe (p = 0.0003, n = 6). Error bars, S.E.