E021020 01

Primary antibodies used for Western blot and immunofluorescence were as follows: mouse anti-γH2AX (05-636, Millipore, 1:1000 for immunofluorescence), mouse anti-53BP1 (612523, BD Transduction Laboratories, 1:500 for immunofluorescence), mouse anti-RPA23 (ab2175, Abcam, 1:1000 for Western blot ), rabbit anti-phospho-RPA (ab109394, Abcam, 1:50000 for Western blot ), and mouse anti-β-actin (E021020-01, Earthox, 1:1000 for Western blot). Chemical agents used were as follows: RO3306 (Selleck), CDK2 inhibitor II (Selleck), Aphidicolin (Sigma), camptothecin (Selleck), Cisplatin (Selleck), and Olaparib (Selleck).

Western blot was performed as described previously.^{22 (link)} Hippocampi from both hemispheres were dissected. For Experiment 2, DG and CA1–3 regions were further separated according to an established protocol.³² Hippocampal tissues were homogenized in ice-cold lysis buffer and centrifuged at 10 000 r.p.m. for 20 min at 4 °C. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Samples containing 20 μg of protein were resolved by 10% sodium dodecyl sulfate-polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were labeled with rabbit anti-nectin-3 (1:2000; Abcam), mouse anti-actin (1:10 000; E021020-01, EarthOx, Millbrae, CA, USA) or mouse anti-GFP (1:20 000; sc-9996, Santa Cruz) overnight at 4 °C. Following incubation with horseradish peroxidase-conjugated secondary antibodies (1:2000, Sigma-Aldrich, St Louis, MO, USA) for 3 h at room temperature, bands were visualized using an enhanced chemiluminescence system (Pierce) and quantified with densitometry (Quantity One 4.2, Bio-Rad, Hercules, CA, USA). All results were normalized by taking the value of the control group as 100%. Each assay was repeated for at least three times.

Western blot was performed as described previously [15 (link)]. MEC and hippocampal tissue from both hemispheres was dissected and homogenized in ice-cold lysis buffer and centrifuged at 10,000 rpm at 4 °C (20 min). Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Samples containing 20 μg of protein were resolved by 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were then labeled with primary rabbit anti-nectin1 (1:2000, Santa Cruz), rabbit anti-nectin3 (1:2000, Abcam), mouse anti-actin (1:10,000, E021020-01, EarthOx, Millbrae, CA, USA), mouse anti-beta-tubulin (1:10,000, E021040-01, EarthOx), or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5000, E021010-01, EarthOx) antibodies at 4 °C (overnight). Following incubation with horseradish peroxidase-conjugated secondary antibodies (1:2000, Sigma-Aldrich, St Louis, MO, USA) at room temperature (3 h), bands were visualized using an enhanced chemiluminescence system (Pierce) and quantified by densitometry (Quantity One 4.2, Bio-Rad, Hercules, CA, USA). Each assay was repeated three times. Results were normalized by taking the mean value of the control group as 100%.