Nrf2 antibody

The expression of HACE1, NRF2, and Ki-67 in glioma tissues was evaluated by IHC using HACE1 antibody (Abcam), NRF2 antibody (Santa Cruz Biotechnology, Inc.), and Ki-67 antibody (BD Pharmingen). The detailed protocol was similarly described according to a previous study.³⁸ Sections were then analyzed by a Tissue FAXS system (Tissuegnostics USA, Tarzana, CA, USA), and positive cells were directly counted using HistoQuest cytometry software according to the principle of flow cytometry.^{39 (link)}

ISL (purity > 99%), GA (purity > 99%), and TP (purity ≥ 98%) were purchased from On-Road Biotechnology Co., Ltd. (Changsha, China). tert-butylhydroquinone (tBHQ), dimethyl sulfoxide (DMSO), and methyl thiazolyl tetrazolium (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glutathione (GSH) Detection Kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Reactive Oxygen Species Assay Kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Nrf2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HO-1, NQO1, and MRP2 antibodies were purchased from Abcam Biotechnology Co. (Milton, Cambridge, UK). Other chemicals were of analytical grade from commercial suppliers.

The coumarins ESC (purity: ≥98%), SCOP (≥99%), FRAX (≥98%) and DAPH (≥97%), 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), tert-butyl hydroperoxide solution (t-BuOOH), monochlorobimane (MCB), dihydroethidium (DHE), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 2,2-diphenyl-1-picrylhydrazyl (DPPH), propidium iodide (PI), PD98059 (PD), DL-Buthionine-(S,R)-sulfoximine (BSO) and anti-β-actin antibody were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). LY294002 (LY) was purchased from Alexis Biochemicals (Alexis Biochemicals, San Diego, CA, USA). The Nrf2 antibody was purchased from Santa Cruz (Santa Cruz Biotecnology, Dallas, TX, USA). Akt (serine–threonine kinase), Phospho-Akt, Erk1/2 (extracellular signal-regulated kinase), Phospho-Erk1/2, GSK3β (glycogen synthase kinase-3β), Phospho-GSK3β and Lamin B1 antibodies were purchased from Cell Signaling (Cell Signaling Technology, Danvers, MA, USA). Beta-Amyloid (1-42) peptide was purchased from AnaSpec (AnaSpec, Fremont, CA, USA). The Nuclear Extract and TransAM Nrf2 Kit were purchased from Active Motif (Active Motif, Carlsbad, CA, USA). All chemicals used were of high purity analytical grade.

HUVECs were cultured in confocal dishes (2 × 10⁵ cells/well) and treated with Hcy (1 mmol/L) in the presence or absence of Gas (200, 400, 800 μg/mL) for 24 hours. After the treatment, the cells were washed twice with PBS and then fixed with 4% (w/v) paraformaldehyde for 15 minutes at room temperature. Then, the cells were washed with PBS twice, and subsequently, the cells were permeabilized with 0.3% Triton X‐100 for 10 minutes. Then, 0.03% Triton X‐100 and 5% BSA in PBS were added to the dishes to block for 30 minutes at room temperature. Next, the cells were incubated with Nrf2 antibody (1:100, Santa Cruz, sc‐365949, CA, USA) diluted in PBS containing 0.3% BSA at 4°C overnight. Then, the cells were washed twice with PBS and incubated with secondary antibody (1:500, Abcam, ab150116, Cambridge, UK) diluted in PBS for 2 hours at 37°C. Finally, the images were captured by a laser confocal fluorescence microscope (FV1000, Olympus, Japan) at a thickness of 1 μm.

As described [44 (link), 46 (link)], the Co-IP assay was performed to test the association between Keap1-Nrf2. In brief, OB-6 osteoblastic cells with applied treatment were lysed [44 (link)]. To the cleared lysates, 0.25 μg of Nrf2 antibody (Santa Cruz Biotech) was added per 0.8 mg of total cellular lysate proteins, and the immune complex formed by rotation for 24 hours at 4°C. The protein A/G-Sepharose (25 μL, Sigma) was then added and the incubation continued for additional 12 hours. The resulting immuno-precipitates captured with protein A/G-Sepharose were washed four times with CHAPS-containing buffer and analyzed by Western blotting assay.