Anti nrf2 antibody

Cells (2 × 10⁷) were cross-linked then nuclear lysates were prepared using a Sonication ChIP Kit (ABclonal) to shear DNA to approximately 500 bp. The samples were immunoprecipitated overnight at 4 °C using IgG or anti-NRF2 antibody (Proteintech) and the levels of target genes were determined via RT-qPCR. The primers used in this study are listed in Table S3.

The cells on coverslips were fixed in paraformaldehyde (4%) for 20 min and washed three times with PBS. The cells were reacted with anti-Nrf2 antibody (1:200 dilution, Proteintech) at 4 °C overnight, and then incubated with Cy3-conjugated anti-rabbit IgG (1:100 dilution, ASPEN) for 40 min at 37 °C. After 30 min of nuclei staining with DAPI, the coverslips were examined by fluorescence microscopy (OLYMPUS).

IEC‐6 cells were cultured in 12‐well plates with chamber slides (5 × 10⁴ cells per well) and incubated overnight. After treated with 10a for 1 hour, the cells were irradiated with 6.0 Gy and cultured for another 24 hours. The cells were fixed with 4% paraformaldehyde for 30 minutes and then permeabilized in Triton X‐100 for 15 minutes. After blocked with 5% bovine serum albumin (BSA) for 3 hours, the slides were incubated with anti‐Nrf2 antibody (1:200 dilution, Proteintech) overnight at 4℃ and stained with FITC‐conjugated secondary antibody (1:50 dilution, Proteintech) for 1 hour in dark. The cell nuclei were stained with DAPI‐containing sealing agent and images were taken using a fluorescence microscope (Life Technologies).

Phorbol 12-myristate 13-acetate (PMA), human interferon gamma (IFNγ), lipopolysaccharides (LPS), dimethyl sulfoxide (DMSO), sulforaphane (SFN), dimethyl fumarate (DMF), Oltipraz (OTZ) were purchased from Sigma (MERCK). Wogonin (WG) and anti-GAPDH antibody were obtained from Millipore (MERCK). Dual Luciferase (Firefly-Renilla) Assay System was purchased from BPS Bioscience. RPMI (Roswell Park Memorial Institute) 1640 culture medium, phosphate-buffered saline (PBS), fetal bovine serum, and human GM-CSF were obtained from Eurobio Scientific. Tryptic soy broth and tryptic soy agar were from Conda laboratories (Dutscher). Lipofectamine^® LTX & PLUS^™ Reagent, Live/Dead^™ BacLight^™ bacterial viability kit, CellROX^™ Green Oxidative Stress Reagent, Maxima First Strand cDNA synthesis kit, and Trizol were obtained from ThermoFisher scientific. Fluoromount-G^™ (EMS) was purchased from Euromedex. DC Protein Assay Reagents and iTaq SYBR green supermix were purchased from Bio-Rad. Anti-Lamin A/C antibody was purchased from Abcam. Anti-Nrf2 antibody was obtained from ProteinTech. IRDye^® 680CW goat anti-mouse IgG, and IRDye^® 800CW Goat anti-Rabbit IgG secondary antibodies were purchased from LI-COR^® Biosciences.

Paraffin-embedded liver tissues were sliced into 3-μm sections for hematoxylin and eosin staining. IHC staining with an anti-Nrf2 antibody (1:100, ProteinTech) was performed using the avidin–biotin immunoperoxidase method as previously described [8 (link)].