Protease and phosphatase inhibitors

Cells were collected by trypsinization and lysed in RIPA lysis buffer (150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris base, pH 8.0). Phosphatase and protease inhibitors (Gold Biotechnology) were added freshly to the lysis buffer. Following gel electrophoresis and transfer of cell extracts onto nitrocellulose, membranes were incubated for 1 hr or overnight in blocking buffer (5% milk in TBS + 0.1% tween). Membranes were subsequently incubated with primary antibodies diluted in antibody buffer (3% BSA in TBS + 0.1% tween) for 2 hrs at room temperature or overnight at 4°C. Detection was achieved using appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. Anti-ZRANB3 (1:5,000, Bethyl Laboratories), anti-SMARCAL1 (1:1,000, Santa Cruz Biotechnology), anti-BRCA1 (1:500, Bethyl Laboratories), anti-BRCA2 (1:1,000, Bethyl Laboratories), anti-vinculin (1:100,000, Sigma-Aldrich), anti-β-actin (1:100,000, Novus Biologicals), anti-GAPDH (1:5,000, Novus Biologicals), anti-HLTF (1:2,000, Abcam), anti-LAMIN B1 (Thermo Fisher Scientific), anti-FANCD2 (1:1,000, Novus Biologicals), anti-PCNA (1:100,000, Abcam), anti-NBS1 (GeneTex), anti-MRE11 (Cell Signaling Technology), anti-RPA2 (Bethyl Laboratories) antibodies were used in western blot experiments.