Anti hltf

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.

Cells were harvested, lysed, and processed for western blot analysis as described previously using 150mM NETN lysis buffer (20 mM Tris [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL leupeptin, and 10 mg/mL aprotinin). For cell fractionation, we isolated cytoplasmic and soluble nuclear fractions with the NE-PER Kit (Thermo Scientific) according to the manufacturer’s protocol; to isolate the chromatin fraction, the insoluble pellet was resuspended in RIPA buffer and sonicated in a BioRuptor according to the manufacturer’s protocol (high power, 15 min, 30 s on and 30 s off at 4°C). Proteins were separated using SDSPAGE and electrotransferred to nitrocellulose membranes. Membranes were blocked in 5% milk PBS-Tween and incubated with primary antibody for 1 hr. Abs for western blot analysis included anti-PCNA (Abcam), anti-H2B (Cell Signaling Technology), anti-β-actin (Sigma), anti-FANCJ (E67), anti-HLTF (Abcam), anti-SMARCA1 (Abcam), and anti-HLTF (Santa Cruz Biotechnology). Membranes were washed, incubated with horseradish-peroxidase-linked secondary antibodies (Amersham), and detected by chemiluminescence (Amersham).