Antibodies against nrf2

O-phenylenediamine (o-PD), perchloric acid, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), bovine serum albumin (BSA), MGO, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), catechin, rutin, luteolin, naringenin, and genistein were purchased from Sigma (St. Louis, MO, USA). Daidzein was obtained from LC Laboratories (Wobum, MA, USA). The LLC-PK1 cell line and fetal bovine serum (FBS) were purchased from American Type Culture Collection (Rockville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from HyClone (Logan, Utah, USA). Antibodies against Nrf2, Glo1, RAGE, and α-tubulin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibody against AGE was procured from Abcam (Cambridge, MA, USA), 2-methylquinoxaline (2-MQ), and 5-methylquinoxaline (5-MQ) was purchased from Tokyo Chemical Industries (Tokyo, Japan), while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Pro-Prep^TM were purchased from iNtRON Biotechnology (Seongnam, Korea).

Based on the structure in previous studies [19 (link), 20 (link)] (also see Figure 1A), MIND4-17 was synthesized and verified by Minde Biotech (Suzhou, China). Puromycin was obtained from Sigma-Aldrich (Nanjing, China). The reagents for cell culture were provided by Gibco (Guangzhou, China). Antibodies against Nrf2, HO1, NQO1, GCLM and GAPDH were purchased from Santa Cruz Biotech (Beijing, China). Antibodies for cleaved-caspase-3 and cleaved-PARP were provided by Cell Signaling Tech (Nanjing, China).

Antibodies against Nrf2, phospho-Nrf2, Bax, and Bcl-xL were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). NQO1, lamin A/C, and caspase-3 antibodies were obtained from Cell Signaling (Danvers, MA, USA). Glutamate cysteine ligase (GCL) antibody was purchased from Abcam (Cambridge, UK) and a Bad antibody was purchased from BD biosciences (Franklin Lakes, NJ, USA). MTT, metaphosphoric acid, DCFH-DA, t-butylhydroperoxide (t-BHP), rotenone, dimethylsulfoxide, and β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). siNrf2 was purchased from Dharmacon (Lafayette, CO, USA).

Loliolide (purity: 98% by HPLC) was purchased from Chemfaces (Wuhan, China). HaCaT and B16F10 cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and penicillin-streptomycin were purchased from HyClone (Logan, UT, USA). 3-(4-5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco (Brisbane, Australia). 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), ascorbic acid, α-melanocyte stimulating hormone (α-MSH), TRIzol, and arbutin were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). The cDNA synthesis kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Forward and reverse primers for polymerase chain reaction (PCR) and real-time PCR were synthesized by Macrogen (Seoul, Korea), and PCR premix was purchased from Bio-D Inc. (Seoul, Korea). Polyvinylidene difluoride (PVDF) membrane was purchased from Merck Millipore (Billerica, MA, USA). Antibodies against PI3K, p-PI3K, AKT, p-AKT, Keap1, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against NRF2, HO-1, CREB, p-CREB, MITF, and tyrosinase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Ethanol- or TCS-treated hMSCs were seeded at 2,000 cells per square centimeter on four-well glass chamber slides (Nalge Nunc International, Rochester, NY, USA), and the cells were incubated in a 5% CO₂ incubator at 37 °C. The cells were fixed with 4% paraformaldehyde (Sigma) for 30 minutes, and then permeabilized with 1% Triton X-100 for 10 minutes followed by blocking for 1 hour with 5% bovine serum albumin (BSA) in PBS. The cells were incubated with 1:100 dilution of antibodies against Nrf2 (Santa Cruz Biotechnology) for 4 hours at room temperature, and then incubated fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Santa Cruz Biotechnology) at a 1:5,000 dilution in 1% BSA-containing PBS for 1 hour at room temperature in the dark. The nuclei were stained with 4,6-diamidino-2-phenyindole (DAPI) (Sigma) and then examined using a Zeiss LSM700 scanning laser confocal microscope (Zen 2011; Carl Zeiss MicroImaging GHBH, Jena,Germany).