The crude lysates were prepared with young adult animals in PRO-PREP lysis buffer (iNtRON, Seongnam, South Korea) supplemented with a complete protease inhibitor cocktail (Roche, Basel, Switzerland) by sonication. After centrifugation (11 000 × g, 4 °C, 10 min), the supernatants were collected and protein concentrations were determined by standard Bradford assay (Bio-Rad, Hercules, CA, USA). The lysate containing 2 mg of proteins was used to incubate with 4 μg of RPA194 antibody (sc-28714, Santa Cruz, Dallas, TX, USA) for 1–2 h at 4°C. The resulting immunocomplex was bound and precipitated with 50 μl protein A/G plus agarose beads (Santa Cruz) for 1 h. The beads were washed three times with NET buffer (50 mM Tris HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) and boiled in Laemmli sample buffer for 10 min. After 10 min of centrifugation (12 000 × g, 4°C), lysate suspensions of the respective sample were analyzed in 8% SDS-PAGE. Proteins were transferred to a PVDF membrane and the resulting blots were detected with the primary antibodies as indicated. The DAO-5-specific monoclonal antibody 5E9 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA)21 (link)was used in 5–10 times dilution with 5% skim milk/PBST (0.5% Tween-20). The blots were developed and detected with chemiluminescence ECL kit (T-Pro Biotechnologies, New Taipei City, Taiwan).