Mouse anti 5mc

gDNA samples and in vitro oxidation products were respectively denatured at 99°C for 10 min and placed quickly on ice for 5 min. Denatured gDNA was mixed with ice-cold 20× saline–sodium citrate (SSC) buffer at a final concentration of 4.8× SSC and blotted on a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA), which was pre-equilibrated in 20× SSC. After air-drying, the membrane was blocked with 3% milk in PBST (PBS containing 0.1% Tween) for 30 min at room temperature (RT), followed by incubation with either mouse anti 5mC (1:1000, Eurogentec, Seraing, Belgium) or rabbit anti 5hmC (1:5000, Active Motif, La Hulpe, Belgium) antibodies for 1 h at RT. The membrane was washed 3× for 10 min with PBST, before it was incubated with horseradish peroxidase (HRP)-conjugated anti mouse IgG (1:5000, GE Healthcare, Freiburg, Germany) or anti rabbit IgG (1:5000, Sigma Aldrich, St. Louis, MO, USA) antibody for 1 h at RT. After three washing steps, remaining signals were detected using Amersham ECL detection reagent (GE Healthcare, Freiburg, Germany) and imaged on a Fuji LAS-1000 imager (FUJI Film, Minato, Tokio, Japan).