Total protein extraction and western blot analysis were performed according to our previously described methods [11 (link), 17 (link)] with the following modifications. The total proteins were electrophoresed on Extra PAGE One Precast Gel 5–20% (Nacalai Tesque, Kyoto, Japan) or Super Sep ACE Gel 5–20% (Wako Pure Chemical). The fractionated products were electrophoretically transferred onto Hybond-P membranes (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). Then, the membranes were blocked in Blocking One (Nacalai Tesque) for 60 min at room temperature and incubated with mouse anti-γH2AX monoclonal antibody (JBW301) (Upstate Biotechnology Inc., Charlottesville, VA, USA) or mouse β-actin monoclonal antibody (Sigma-Aldrich). After washing, the membranes were incubated with the anti-mouse IgG HRP-Linked Whole Ab (from sheep) (NA931) (GE Healthcare Bio-Sciences Corp.) for 60 min at room temperature. Immunoblotting was performed using Select Western Blotting Detection System (GE Healthcare Bio-Sciences Corp.). Protein bands were visualized using ChemiDoc XRS system (Bio-Rad).