Monoclonal anti nrf2

AA was purchased from Shanghai Nature Standard R&D and Biotech Co., Ltd. (purity 98.0%; molecular weight 488.70; Shanghai, China). Carbon tetrachloride (CCl₄) was purchased from Shenzhen Xunye Chemical (Shenzhen, China). The biochemical kits of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were purchased from Jiancheng Institute of Biotechnology (NanJing, China). The trizol reagent, primescript RT reagent, and real time-PCR kit were purchased from TaKaRa (Dalian, China). Monoclonal anti-Nrf2, anti-HO-1, anti-NQO-1, anti-GCLC, histone H3, and β-actin antibodies were obtained from Abcam (Cambridge, MA, USA). Monoclonal antibodies against NF-κB, IκBα, JAK1, phospho-JAK1, STAT3, and phospho-STAT3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies used in Western blotting were goat anti-rabbit IgG (H+L) (Bioworld Technology, Shanghai, China). Secondary antibodies used in immunohistologic staining were purchased from Dako (Glostrup, Denmark). Secondary antibodies used in immunofluorescence staining were purchased from Abcam.

Cells were lysed and nuclear and cytosolic extracts were prepared as described22 and tissue total lysates were prepared using the inflamed distal colon.20 Protein concentrations in the lysates were determined by the bicinchoninic acid assay. Cell or tissue extracts were subjected to Western blot analysis.20 Nrf2, p65, and HIF-1α were detected in nuclear extracts (30–40 µg) using monoclonal anti-Nrf2 (Abcam, Cambridge, MA, USA), p65 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), and HIF-1α (BD Biosciences, San Jose, CA, USA) antibodies, and hemeoxygenase-1 (HO-1) protein was detected in whole cell (30–40 µg) or tissue lysates (30–40 µg) using monoclonal anti-HO-1, COX-2, and iNOS antibodies (Santa Cruz Biotechnology). Secondary antibodies (Santa Cruz Biotechnology) for each primary antibody were used at a dilution of 1:2,000. SuperSignal chemiluminescence substrate (Pierce, Rockford, IL, USA) was used for visualizing signals. Experiments were performed in duplicate and normalized with antibodies to topoisomerase II (Santa Cruz Biotechnology) for transcription factors and to α-tubulin (Santa Cruz Biotechnology) for HO-1, COX-2, and iNOS.