Figure 5.

Cooperative binding of JDP2, MafK and Nrf2 to the ARE. (a) Tentative location of cis-elements in the ARE promoter region and its deletions mutants used for EMSA assay. AP-1-like (nt −471 to −466), ARE core (−462 to −456) and GC box (−455 to −453) elements are shown in white rectangular boxes. The deleted elements are shown in black rectangular boxes. The nucleotide sequences of the mutants (M1 to M7) were listed in the Supplementary Table I. (b) Competitive EMSA assay of JDP2 with ARE mutants. EMSA reactions were performed in the presence or absence of a competitive ARE as described in Materials and Methods, using [γ-³²P]-labeled double-stranded ARE oligonucleotides. GST–JDP2 and GST proteins were purified using GST affinity resins. (c) Competitive EMSA assay of MafK with ARE mutants. IVT–MafK proteins were incubated with an ARE probe in the presence or absence of competitive ARE mutants and EMSA assay was performed as described in Materials and Methods. IVT alone was used as a negative control. (d) Supershift EMSA assay with nuclear extracts (NEs) from WT MEFs and Jdp2 KO MEFs using the DNA probe of human NQO1–ARE. NEs from WT MEFs and Jdp2 KO MEFs were incubated without (lanes 1 and 6) and with antibodies specific for Jdp2 (lanes 2 and 7), Nrf2 (lanes 3 and 8), MafK (lanes 4 and 9) and IgG (lane 5 and 10). ‘Bound' indicates the supershifted DNA–protein complexes and ‘Free' indicates the ARE–DNA probe