Ab96488

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).

Western blots were imaged on a FUSION SOLO (Witec AG) chemiluminescence imaging system. The following primary antibodies (at a dilution of 1:1000) were used: β-actin-horseradish peroxidase (HRP) (C4, sc-47778; Santa Cruz Biotechnology), DNA2 (ab96488; Abcam), CIAO1 (ab83088; Abcam), MIP18/FAM96b (20108-1-AP; Proteintech), MMS19 (16015-1-AP; Proteintech). Secondary antibodies (at a dilution of 1: 2500) were anti-Mouse-HRP (NA931; Amersham) and anti-Rabbit-HRP (NA934; Amersham). Uncropped blots can be found in Supplementary Fig. 6.