Calcium and magnesium free phosphate buffered saline

Bovine aortic endothelial cells were purchased from Cell Applications (San Diego, CA, USA). The following materials were purchased from the indicated vendors: Dulbecco's modified Eagle's medium and calcium-and magnesium-free phosphate buffered saline from Nissui Pharmaceutical (Tokyo, Japan); fetal bovine serum, Opti-MEM ® Reduced Serum Medium, Lipofectamine ® RNAiMAX Transfection Reagent, and High-Capacity cDNA Reverse Transcription Kit from Thermo Fisher Scientific (Waltham, MA, USA); cell culture dishes and plates from Corning (Corning, NY, USA); QIAzol lysis reagent from QIAGEN (Venlo, The Netherlands); GeneAce SYBR ® qPCR Mix α from Nippon Gene (Tokyo, Japan); and 1,3-diaminopropane from Tokyo Chemical Industry (Tokyo, Japan). Other reagents were purchased from Nacalai Tesque (Kyoto, Japan).

Cu10 was purchased from Tokyo Chemical Industry (Tokyo, Japan). Bovine aortic endothelial cells were purchased from Cell Applications (San Diego, CA, USA). Dulbecco's modified Eagle's medium and calcium-and magnesium-free phosphate buffered saline were from Nissui Pharmaceutical (Tokyo, Japan). Fetal bovine serum was from HyClone Laboratories (Waltham, MA, USA). Bicinchoninic acid protein assay reagent kit was from Thermo Fisher Scientific (Waltham, MA, USA). Polyvinylidene difluoride membrane (0.2 μm), sodium diethyldithiocarbamate trihydrate (Na01), and mouse monoclonal anti β-actin antibody were from Wako Pure Chemical Industries (Osaka, Japan). Mouse monoclonal anti-MT-1/2 antibody (E9) was from Dako (Glostrup, Denmark). Rabbit polyclonal anti-human Nrf2 antibody (H-300) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated anti-rabbit IgG antibody (#7074) and horseradish peroxidase conjugated anti-mouse IgG antibody (#7076) were from Cell Signaling (Beverly, MA, USA). High-Capacity cDNA Reverse Transcription kit was from Applied Biosystems (Foster, CA, USA). Gene Ace SYBR qPCR Mixα was from Nippon Gene (Tokyo, Japan). Dual-Luciferase Reporter Assay System and pRL-SV40 were from Promega (Madison, WI, USA). Chemi-Lumi One L and other reagents were from Nacalai Tesque (Kyoto, Japan).