Pnrf2

Before transferring the proteins to a PVDF membrane, the prepared protein samples were loaded into a 10% acrylamide gel for western blotting electrophoresis and blocked with a 5% skim milk solution. The membrane was incubated in primary antibody diluted with Tris-buffered saline (TBS) supplemented with 0.05% Tween (TBS-T) for 2 h. After three washes with TBS-T, the diluted secondary antibody was incubated for 1 h. Detection was performed after the addition of the luminol mixture (TLP-112.1; Translab, Korea). The following primary antibodies were used for western blotting: ATG5 (sc-515347, Santa Cruz, USA), p21 (sc-6246, Santa Cruz, USA), MyoD (sc-377460, Santa Cruz, USA), myogenin (sc-12732, Santa Cruz, USA), MYH (sc-376157, Santa Cruz, USA), LC3 (2775S, Cell Signaling Technology, USA), mTOR (2972S, Cell Signaling Technology, USA), p-mTOR (2974T, Cell Signaling Technology, USA), eIF2-α (9722S, Cell Signaling Technology, USA), p-eIF2α (9721S, Cell Signaling Technology, USA), Nrf2 (sc-365949, Santa Cruz, USA), p-Nrf2 (PA5-67520, Invitrogen, USA), β-actin (sc-47778, Santa Cruz, USA) and α-tubulin (sc-8035, Santa Cruz, USA). For secondary antibodies, we used horseradish peroxidase (HRP)-conjugated goat antibody against rabbit IgG (PA489724, CusaBio, USA) and HRP-conjugated goat antibody against mouse IgG (PA644737, CusaBio, USA).

The reagent contained mangiferin, doxorubicin, dimethyl sulfoxide (DMSO), SDS, tris, glycine, BSA, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenylTrazolium Bromide (MTT), and CelLytic (Sigma-Aldrich, St. Louis, MO, USA). Other ingredients included Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), antimycotic/antibiotic mixtures (Carlsbad, CA, USA), DCFH2-DA, and TUNEL staining kits (Roche, Mannheim, Germany), and antibodies were purchased from Invitrogen, Carlsbad, CA, USA. Antibodies to AKT, pPI3K, PI3K PERK1/2, ERK1/2, pP38, P38, BCL2 PARP, HO-1, NQO1, Keap-1, pNrf2, COX, TNF-α, pNFC-B, β-actin, and Lamin B1 were sourced from Invitrogen and Thermo Fisher, Waltham, MA, USA. The antibody against cleaved caspase-3 was purchased from Abcam (Branford, CT, USA).