Anti 8 ohdg

Protein was extracted using protein extraction solution (PRO-PREP, iNtRON Biotechnology, Kyungki-Do, Korea). The protein, 20 µg, was electrophoresed on a 10% polyacrylamide gel, and transferred to a nitrocellulose membrane (Atoh, Tokyo, Japan). The membranes were reacted overnight at 4°C with the primary antibodies. The primary antibodies applied were anti-ATP5A (Proteintech), anti-8-OHdG (ABBIOTEC, San Diego, CA), anti-XIAP (Abcam), anti-cleaved-caspase-3 (Abcam), or anti-caspase-9 (Proteintech) at a concentration of 0.5, 2.5, 1.0, 1.0, or 1.0 µg/ml. After the incubation with peroxidase-labeled goat anti-mouse or anti-rabbit IgG antibody (Dako Cytomation) for 1 hour at room temperature and vigorous washing, the nitrocellulose membrane was incubated with Chemiluminescence Luminol Reagent (Immuno Star LD, Wako, Tokyo, Japan) and photographed digitally using ImageQuant LAS 4000 mini (GE healthcare Japan Co, Tokyo, Japan). Immunoblot using anti-actin monoclonal antibody (Sigma Chemical Co. St. Louis, MO) was used for standardization. Intensity was measured using the Multi Gauge v3.1 (Fujifilm, Tokyo, Japan). Experiments were repeated at least three times.

For quantification, immunoblotting for TFAM, 8-OHdG and HIF-1α was performed on MLO-Y4 cells. Proteins were extracted using protein extraction solution (PRO-PREPTM, iNtRON Biotechnology, Kyungki-Do, Korea). Extracted protein (20.0µg) was applied to and electrophoresed on a 10% polyacrylamide gel, and transferred to a nitrocellulose membrane (WAKO, Tokyo, Japan). The membranes were reacted overnight at 4 °C with anti-TFAM (RayBiotech, Norcross Georgia, U.S.A), anti-8-OHdG (ABBIOTEC, San Diego, CA), anti-HIF-1α (abcam) at a concentration of 1.0, 2.5, or 1.0 µg/ml. After incubation with peroxidase-labeled goat anti-rabbit or mouse IgG antibody (Dako Cytomation, Tokyo, Japan) for 1 hour at room temperature and vigorous washing, the nitrocellulose membrane was incubated with Chemiluminescence Luminol Reagent (Immuno Star LD, Wako, Tokyo, Japan) and photographed digitally using ImageQuant LAS 4000 mini (GE healthcare Japan Co, Tokyo, Japan). All samples were standardized by immunoblot using anti-actin mouse monoclonal antibody (Sigma Chemical Co., St. Louis, MO).