Anti mouse or anti rabbit horseradish peroxidase conjugated antibody

A DNA dot-blotting was performed using extracted genomic DNA from a DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). The DNA concentration were measured by a microplate reader (Epoch, BioteK, Winooski, VT, USA) at 260 nm/280 nm absorbance. Purified DNA were loaded onto 0.2 µM nitrocellulose membrane and hybridized for 2 h at 80 °C. The membrane was then blocked with 5% skim milk in 1X Tris-buffered saline (Bio-Rad) with 0.1% Tween 20 (TBST; Sigma-Aldrich) at room temperature for 1 h. After incubation with dsDNA (1:2000, Abcam) and 8-OHdG (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C for overnight, membrane was washed in three changes of TBST for 15 min and then incubated with an anti-mouse or anti-rabbit horseradish-peroxidase-conjugated antibody (1:2500, Abcam) at room temperature for 2 h. Dot blots were visualized using electrochemiluminescence (Bio-Rad, Hercules, CA, USA) and evaluated with an Amersham Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden).

Genomic DNA was extracted and isolated from the L4–5 and L5–6 discs in each group using a DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) to quantify 5mC and 5hmC. Purified gDNA was spotted on a nitrocellulose membrane (0.2 µm pore size) and hybridized to the membrane by baking at 80 °C for 2 h. The membrane was then blocked with 5% skim milk and incubated with mouse anti-dsDNA (1:2000, Abcam), mouse anti-5mC (1:100, Active Motif), and mouse anti-5hmC (1:500, Active Motif) at room temperature for 1 h. Samples were then incubated with an anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibody (1:1000, Abcam) for 1 h at room temperature. Antibody binding was visualized using enhanced chemiluminescence.