Sc 376377

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).

Cell lysis was carried out in urea buffer (9 M urea, 50 mM Tris HCL, pH 7.3, 150 mM β-mercaptoethanol) followed by sonication using a soniprep 150 (MSE) probe sonicator. In some instances, cells were lysed in SDS loading buffer (2% SDS, 10% (v/v) glycerol, 2% 2-Mercaptoethanol and 62.5 mM Tris-HCl, pH 6.8) followed by boiling for 10 min. Samples were resolved by SDS-PAGE and transferred to PVDF or nitrocellulose. Protein concentrations were determined by Bradford assay by spectrophotometry using a NanoDrop 2000 device (Thermo Scientific). Immunoblots were carried out using the indicated antibodies: α-Tubulin (Sigma, B-5-1-2; T5168, 1:100,000), BRCA1 (Millipore, OP-92, 1:1000), BRCA2 (Millipore, OP-95, 1:1000), EXD2 (Sigma, HPA005848, 1:1000), MCM2 (Abcam, ab4461, 1:10,000), MRE11 (Abcam, ab214, 1:1000), PCNA (Santa-Cruz, PC-10, 1:500), RECQ1 (Santa Cruz, sc-166388,1:1000) and SMARCAL1 (Santa Cruz, sc-376377 1:1000).

Cells were grown on coverslips or μ-slides (Ibidi) to subconfluence and immunofluorescence FISH (IF-FISH) was performed as previously described^{69 (link)}, using the primary antibodies against SMARCAL1 (mouse monoclonal, sc-376377, Santa Cruz Biotechnology, 1:100) and PML (rabbit polyclonal, ab53773, Abcam, 1:200) in blocking solution (1 mg/mL BSA, 3% goat serum, 0.1% Triton X-100, 1 mM EDTA) overnight at 4 °C. Briefly, cells were fixed with 2% formaldehyde. After washing with phosphate-buffered saline (PBS), slides were incubated with goat secondary antibodies against rabbit or mouse IgG, then conjugated with Alexa Fluor 488 or 594 (ThermoFisher, 1:100) in blocking solution. After washing with PBS, cells were fixed again with 2% formaldehyde for 10 min, and washed once again with PBS. Cells underwent a dehydration series (70%, 95%, 100% ethanol), and then incubated with PNA probes (each 1:1000) TelC-Cy3 and Cent-FAM (PNA Bio) in hybridizing solution, denatured at 70 °C for 5 min on a ThermoBrite system, then incubated in the dark for 2 h at room temperature. Slides were then washed with 70% formamide 10 mM Tris-HCl, PBS, and then stained with 4',6-diamidino-2-phenylindole (DAPI) and sealed.