Figure 7
Validation of newly identified Nrf2-responsive genes by electrophoretic mobility shift assay. Nuclear extract from small airway epithelium was incubated with 32P-labeled antioxidant response element (ARE) oligonucleotides and specifically competed with a fifty-fold excess of ARE oligonucleotides from the same gene or with ARE oligonucleotides from putative Nrf2-modulated genes. (A) NAD(P)H dehydrogenase, quinone 1 (NQO1) ARE, located at −386 bp upstream of the transcription site. Lane 1, no protein; lane 2, nuclear protein alone; lane 3, nuclear protein + specific competitor (NQO1 unlabeled ARE); lane 4, nuclear protein + nonspecific competitor (von Willebrand (vWF) oligonucleotides); lane 5, no protein; lane 6, nuclear protein alone; lane 7, nuclear protein + anti-Nrf2 antibody; lane 8, nuclear protein + IgG control; lane 9, no protein; lane 10, nuclear protein alone; lane 11, nuclear protein + specific competitor (NQO1 unlabeled ARE); lane 12, nuclear protein + pirin (PIR) unlabeled ARE at position + 33 bp (downstream of the transcription start site); lane 13, nuclear protein + PIR unlabeled ARE at position -3209 bp (upstream of the transcription start site); lane 14, nuclear protein + PIR unlabeled ARE at position −3480 bp; lane 15, nuclear protein + PIR unlabeled ARE at position −5566 bp; lane 16, nuclear protein + nonspecific competitor (vWF oligonucleotides); lane 17, no protein; lane 18, nuclear protein alone; lane 19, nuclear protein + specific competitor (NQO1 unlabeled ARE); lane 20, nuclear protein + ABCB6 unlabeled ARE at position −7575 bp; lane 21, nuclear protein + nonspecific competitor (vWF oligonucleotides); lane 22, no protein; lane 23, nuclear protein alone; lane 24, nuclear protein + specific competitor (NQO1 unlabeled ARE); lane 25, nuclear protein + UGT1A4 unlabeled ARE at −3043 bp upstream of the transcription start site; lane 26, nuclear protein + UGT1A4 unlabeled ARE at position −3159; lane 27, nuclear protein + UGT1A4 unlabeled ARE at position −3885 bp; lane 28, nuclear protein + UGT1A4 unlabeled ARE at position −5523 bp; lane 29, nuclear protein + nonspecific competitor (von Willebrand factor (vWF) oligonucleotides). For lane 13, lane 15, lane 27, and lane 28, disappearance of gel-shift band indicates functional ARE. (B) Pirin (PIR) ARE. Radioactively labeled PIR-ARE at position −3209 bp (lane 30−37). Lane 30, no protein; lane 31, nuclear protein alone; lane 32, nuclear protein + specific competitor (PIR unlabeled ARE); lane 33, nuclear protein + nonspecific competitor (vWF oligonucleotides); lane 34, no protein; lane 35, nuclear protein alone, lane 36, nuclear protein + anti-Nrf2 antibody; lane 37, nuclear protein + IgG control; (C) radioactively labeled UGT1A4-ARE at position −3885 bp (lane 38−45). Lane 38, no protein; lane 39, nuclear protein alone; lane 40, nuclear protein + specific competitor (UGT1A4 unlabeled ARE); lane 41, nuclear protein + nonspecific competitor (vWF oligonucleotides); lane 42, no protein; lane 43, nuclear protein alone, lane 44, nuclear protein + anti-Nrf2 antibody; lane 45, nuclear protein + IgG control.