FIGURE 3.

Antioxidant increases binding of Nrf2 to Bcl-2 gene AREr3. A, ChIP assay. Hepa-1 cells were treated with 50 μm t-BHQ for 4 h, fixed with formaldehyde, and cross-linked, and the chromatin was sheared. The chromatin was immunoprecipitated with anti-Nrf2 antibody or control IgG. Nrf2 binding to Bcl-2 promoter was analyzed by PCR with specific primers for the ARE-r3 region of Bcl-2 promoter. GAPDH primers were used as a control. The AREr3 region of Bcl-2 promoter was amplified from 5 μl of purified soluble chromatin before immunoprecipitation to show input DNA (upper two gels). The effect of t-BHQ on the relative binding of Nrf2 to the human NQO1 gene ARE was also determined by the immunoprecipitation of sheared chromatin from HepG2 cells treated with DMSO and t-BHQ followed by a ChIP assay (lower gel). The relative binding of Nrf2 to the Bcl-2 AREr3 and hNQO1 ARE promoter was quantified from the band intensities of three independent experiments and plotted (lower panel). B, ChIP and quantitative (q) RT-PCR. The chromatin was immunoprecipitated as described in A from Hepa-1 cells. The binding of Nrf2 to the AREr3 region of Bcl-2 promoter was measured by quantitative real time PCR using custom-made probes and primers obtained from Applied Biosystems (ID 186710084_1). The mixture was run on a 7500 Real-Time PCR System (Applied Biosystems) using relative quantitation according to the manufacturer's protocols. The amounts of immunoprecipitated DNA were normalized to the inputs and plotted. C, gel and supershift analysis. Bcl-2 AREr3 was end-labeled with [γ-³²P]ATP and T4 kinase. Labeled DNA (100,000 cpm) was incubated with 10 μg of Hepa-1 nuclear extract in binding buffer (lanes 2–5). The nuclear proteins binding to AREr3 were either competed with cold AREr3 (lane 3) or supershifted with IgG (lane 4) and Nrf2 antibody (ab) (lane 5). The reaction mixtures were run on a polyacrylamide gel and autoradiographed. SB, shifted band; SSB, supershifted band. All experiments were performed three times, and one set of data is presented. Error bars indicate S.E. of triplicate samples.