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. 2013 Sep 24;123(10):4195–4207. doi: 10.1172/JCI62891

Figure 6. Identification of a SALL4 DNA binding site in the promoter region of HOXA9 by EMSA assays.

Figure 6

(A) Human HOXA9 promoter region. Corresponding locations of amplicons for qPCR and oligo probes for EMSA are indicated. Left: 3 probe sequences. Right: Mutant probe sequences of probe 3. (B) EMSAs were performed to identify the SALL4 DNA binding cells to the HOXA9 promoter region using nuclear extracts from 293T cells transfected with a SALL4 expression construct. Of 3 pairs of oligonucleotide sequences, only probe 3 was specifically bound by SALL4 protein (lane 8); however, this shift could be prevented by competition from 200-fold excess of nonlabeled probe (lane 9). (C) Endogenous SALL4 from THP1 nuclear extracts bound probe 3 specifically (lanes 1–3). Lanes 4–6 show nuclear extracts from 293T-SALL4 as a positive control. (D) SALL4 antibody abolished SALL4 and probe 3 binding (lane 4), but not IgG control antibody (lane 5). (E) Among the 5 mutant probes, only mutant probe 2 (lane 5) failed to inhibit wild-type probe 3 binding to SALL4 protein.