Figure 5.

EMSA assay for the HRE binding activity of HIF-1α⁴¹⁷ and ARNT. Binding to a radiolabeled oligonucleotide containing the HRE core sequence was observed in nuclear extracts prepared from HEK293 cells transfected with pARNT plus pHIF417 or pHIF-1α. For supershift analysis, 1 μl of ARNT, HIF-1α⁴¹⁷ or HIF-1α-ODDD anti-serum was added to the completed EMSA reaction mixture. 1, DNA binding by HIF-1α⁴¹⁷–ARNT co-expression; 2, supershift of ‘band 1’ by anti-ARNT antibody; 3, supershift of ‘band 1’ by anti-HIF-1α⁴¹⁷ antibody; 4, DNA binding by HIF-1α–ARNT co-expression; 5, supershift of ‘band 4’ by anti-ODDD antibody. The EMSA film was exposed for a longer time to make denser 4 and 5 bands (the right panel).