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. Author manuscript; available in PMC: 2013 Oct 9.
Published in final edited form as: Biochim Biophys Acta. 2012 Feb 9;1823(4):889–899. doi: 10.1016/j.bbamcr.2012.01.017

Fig. 7.

Fig. 7

Binding of HIF-1α to the HRE1 site within the hMSH3 promoter region in hypoxic wt-p53 HCT116+5 and mut-p53 SW620 cells. A and D: Two binding products, “a” and “b”, are formed between the protein complexes containing HIF-1α, A and B respectively, and the radio-labeled (hot) oligonucleotide probe containing the HRE1 site and its flanking sequences (WT-HRE1) in hypoxic wt-p53 HCT116+5 (A) or mut-p53 SW620 (D). –: nuclear extracts were not added (lane 1), NE: nuclear extracts, N: normoxic cell nuclear extracts were added (lanes 2), H: hypoxic cell nuclear extracts were added (lanes 3–8). Left panel: a non-radio-labeled (cold) WT-HRE1 probe was not added (–, lanes 2, 3) or was added 25-fold excess (25, lane 4) or 50-fold excess (50, lane 5) of hot probe. Right panel: anti HIF-1α antibody was either not added (–, lane 6) or added (anti HIF-1α antibody, lane 7), or control mouse IgG (IgG, lane 8) was added to the reaction mixture. F: free probe. Bands “a” and “b” were diminished by the anti HIF-1α antibody. B and E: Inhibition of binding of A or B to hot WT-HRE1 probe by 8-fold excess of cold-WT-HRE1: (diminished bands “a” and “b” in lane 3) in hypoxic wt-p53 HCT116+5 (B) or mut-p53 SW620 (E). No inhibition of binding of A or B to hot WT-HRE1 probe by 8-fold excess of cold-MT1-HRE1 probe that contain exact the same DNA sequence as WT-HRE1 probe except HRE1 site was replaced by oligonucleotides, TTTTG, (lane 5). Inhibition of binding of A but not B to hot WT-HRE1 probe by 8-fold excess of cold-MT2-HRE1 probe containing replaced oligonucleotides, GCATA: (diminished band “a” but not “b” in lane 4). NE: nuclear extracts, H: hypoxic cell nuclear extracts, (+): added to the reaction mixture, (–): not added, F: free probe. These results indicate that the protein complexes A and B specifically bound to the HRE1 site but A has an additional binding specificity to another sequences compared to B. C and F: Inhibition of binding of A or B to hot WT-HRE1 probe by 100-fold excess of cold-HRE2: (diminished bands “a” and “b” in lane 2) in hypoxic wt-p53 HCT116+5 (C) or mut-p53 SW620 (F). No inhibition of binding of A or B to hot WT-HRE1 probe by 100-fold excess of cold-MT1-HRE2 probe that contain exact same sequence as WT-HRE2 probe except HRE2 site was replaced by oligonucleotides, TTTTG, (lane 3). NE: nuclear extracts, H: hypoxic cell nuclear extracts, (–): not added, F: free probe. The results indicate that A and B bound to the HRE2 sites of WT-HRE2 probe.