Sc 98

Antigen retrieval for spinal cord sections was carried out in citrate buffer (0.01 M, pH 6.0) for 40 min at 99°C in a water bath. Endogenous peroxidase activity was inhibited by incubating in 3% H₂O₂ solution for 10 min at RT. Non-specific binding sites were blocked using normal goat serum (AR0009, Boster) for 30 min at RT. Sections were thereafter incubated with rabbit polyclonal anti-glial fibrillary acidic protein (GFAP, 1:500, BA0056, Boster), mouse monoclonal anti-βIII-tubulin (1:200, sc-80016, Santa Cruz), mouse monoclonal anti-cell adhesion molecule L1 (1:200, MAB777, R&D Systems), rabbit polyclonal anti-mTOR (1:200, sc-8319, Santa Cruz), mouse monoclonal anti-p53 (1:1000, sc-98, Santa Cruz), or mouse monoclonal anti-PTEN (1:100,sc-7974, Santa Cruz) antibodies overnight at 4°C. After washing in PBS three times for 5 min each at RT, sections were visualized with a DAB Detection Kit (PV-9000-D, ZSGB-Bio, Beijing, China) and then stained in the dark using a 3-amino-9-ethylcarbazole (AEC) kit (0037, Maixin Biotech, Fuzhou, China) according to the manufacturer's protocols. Stained sections were mounted in water-soluble mounting medium (AR1018, Boster). IHC images were taken under a light digital microscope (DM-117M, Jiangnan, Jiangsu, China) 24 h after the slides were mounted.

Equal amounts of protein samples (whole-cell lysate, subcellular fractions, or IP samples) were heated at 70 or 100 °C for 10 min, run on a precast Tris-acetate (for BRCA2 blots; EA03752BOX, Thermo Fisher Scientific) or Mini-PROTEAN® TGX™ protein gel (Bio-Rad) and then transferred to a nitrocellulose membrane (162-0145, Bio-Rad). The membrane was blocked in 5% non-fat dry milk in PBST (PBS with 0.05% Tween-20) and incubated overnight with primary antibodies at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature. Uncropped images of western blots are shown in Supplementary Fig. 15.
Primary antibodies used were BRCA2 (1:300; OP95, EMD Millipore), clathrin (1:3000; 610499, BD Biosciences), DNA2 (1:500; ab96488, Abcam), EXO1 (1:1000; A302-640A-T, Bethyl Laboratories), FLAG (1:1000; A8592, Sigma), MRE11 (1:5000; a gift from Dr John Petrini), PARP1 (1:1000; sc-7150, Santa Cruz Biotechnology), p53 (1:1000; sc-98, Santa Cruz Biotechnology), p21 (1:1000; sc-6246, Santa Cruz Biotechnology), HDAC2 (1:2000; 2540S, Cell Signaling Technology), SMARCAL1 (1:500; sc-376377, Santa Cruz Biotechnology), tubulin (1:10,000; T9026, Sigma), RAD51 (1:2000; PC130, EMD Millipore), histone H3 (1:2000; 9715, Cell Signaling Technology). Secondary antibodies used were peroxidase-linked anti-mouse or anti-rabbit IgG (1:10,000; GE Healthcare).

Antibodies were purchased from the following suppliers: anti-H3K9me2, anti-H3K27me3 and anti-H3K9ac (EpiGentek, Farmingdale, NY); anti-H3K4me3, anti-H3K4me1 and anti-β-actin (Abcam, Cambridge, UK); negative rabbit IgG (Cell Signaling Technologies); anti-PRDM1 (sc-66015, Santa Cruz Biotechnologies, Dallas, TX); anti-TP53 (P6874 from Sigma and sc-98 from Santa Cruz); anti-mouse HRP-conjugated and anti-tubulin (Sigma); and anti-FOXA1 from Santa Cruz. Recombinant human TP53 protein (ref. 506165) was obtained from Calbiochem (San Diego, CA). The inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) was purchased from Sigma-Aldrich and dissolved in DMSO.