Fig 1: Nrf2 and MafG occupy and transactivate the MARE-1 element at proximal promoter region of AHSP.(A) JASPAR analysis predicted a putative MafG/K binding site, MARE-1, located +304 to +315 bp downstream of the AHSP transcriptional initiation site. Sequences of the original MARE-1 and the mutant used in (F) are shown.(B) ChIP-seq data showing MafG and MafK binding status in the proximal promoter region of AHSP in K562 cells (data obtained from MafG: GSE92076 and MafK: GSE31477).(C and D) EMSA of the MARE-1. A probe containing the MARE-1 was incubated with purified MafG protein (C) or purified Nrf2 and MafG proteins (D). Excess unlabeled cold probe (200-fold) was included as indicated.(E) ChIP analysis of Nrf2, MafG and MafK occupancy at the MARE-1 site on the AHSP gene in a-globin-overloaded and control K562 cells (the data are presented as the mean ± SD; ns, Not significant; **, P < 0.01; n = 3 replicates).(F) Dual-luciferase reporter assays to determine the enhancer activity of the MARE-1-WT fragment (+52 to +552 bp from the AHSP transcriptional initiation site), the effect of the MARE-1 element mutation, and the ability of Nrf2 to transactivate the enhancer activity of the MARE-1-WT fragment. The pGL3-promoter vector with the SV40 promoter was used as a control (the data are presented as the mean ± SD; **, P < 0.01; ***, P < 0.005; n = 3 replicates).(G) Dual-luciferase reporter assays to determine the ability of MafK or MafG to cooperate with Nrf2 in transactivating the enhancer activity of the MARE-1-WT fragment (the data are presented as the mean ± SD; ns, ***, P < 0.005; n = 3 replicates).
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