Chromatin immunoprecipitation (ChIP) assay

Cells were treated with 400 µM H₂O₂ in the presence or absence of 500 µg/ml LBP for 24 h. Subsequently, a ChIP assay was conducted to examine the binding of Nrf2 in HO-1 promoters. Following the experiment, cells were washed, fixed in 1% formaldehyde for 10 min at room temperature and sonicated (on 3 sec and off 10 sec for 10 times at room temperature) to shear chromatin. Nrf2 antibody (dilution 1:50) was added to the cleared lysate and binding was allowed to proceed at 4°C overnight. The complex was eluted and the released DNA was amplified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) as stated below.