FIGURE 2:

Nrf2 binds the human p66Shc promoter containing the ARE and AP1-like sequences. (A) K562 cells were treated with 20 μM hemin for 36 h, and nuclear (Nuc.) and cytoplasmic (Cyto.) fractions were used for Western blot with Nrf2, lamin B1, or LDH antibody. Lamin B1 and LDH are nuclear and cytoplasmic fraction markers, respectively. (B) Schematic of the 5′-promoter region of the human p66Shc gene, which contains an ARE and an AP1-like sequence. n = any base; M = A or C; R = A or G; Y = C or T. Asterisks indicate matched bases. (C) K562 cells treated with 0, 20, 40, and 75 μM hemin for 12 h were subjected to ChIP assays with control IgG or Nrf2 antibody, followed by real-time PCR using p66Shc primers spanning the ARE, AP1-like, a 2.3-kb upstream 5′-promoter region, and a β-globin promoter region. Data were normalized by input DNA and are shown as mean ± SD (n = 3). (D) Nuclear extracts of K562 cells treated with 20 μM hemin for 36 h were used for pull-down assay. Nrf2 binding to a biotinylated double-strand p66Shc ARE (−107 to −82 base pairs) or a p66Shc AP1-like (−291 to −266 base pairs) probe was detected by Western blot using an Nrf2 antibody. A human ferritin H ARE (–4433 to –4412 base pairs) probe was used as a positive control and no DNA probe in the binding reaction as a negative control. Coomassie brilliant blue (CBB) staining is shown for verification of equal loading. (E) K562 cells were transfected with wild-type or mutant pGL3 p66Shc-450/+60 plasmid and incubated with 0, 20, and 40 μM hemin for 24 h. Firefly luciferase activity was normalized by Renilla luciferase activity and presented as relative to nontreated wild-type p66Shc-450/+60 plasmid. Top, asterisks indicate mutated nucleic acid. Bottom chart, asterisks indicate significantly different from nontreated cells transfected with wild-type p66Shc-450/+60 plasmid. *p < 0.005, **p < 0.001 (n > 3).