Immunoprecipitation experiments were performed as previously described (25 (link)). Lysates were prepared from 2.5 × 106 cells using RIPA buffer (0.1% SDS, 0.5% Na-deoxycholate, 1% NP40, 150 mM NaCl, 1 mM EDTA, 50 mM Tris/Cl, pH 8) supplemented with phosphatase, protease inhibitors and benzonase. One milligram of lysate was incubated overnight at 4°C with BcMagTM Magnetic Beads (Bioclone) conjugated with 4 μg of anti-RECQ1 antibody under rotation, according to the manufacturer instructions. After extensive washing in RIPA buffer, proteins were eluted in 2× electrophoresis buffer and subjected to SDS–PAGE and western blotting.
Western blotting were performed using standard methods. Blots were incubated with primary antibodies against RECQ1 (Santa Cruz Biotechnology), SMARCAL1 (Bethyl), MRE11 (Novus Biological), DNA2 (Abcam), EXO1 (Santa Cruz Biotechnology), anti-PAR (Abcam), tubulin (Sigma-Aldrich) and lamin B1 (Abcam). After incubations with horseradish peroxidase-linked secondary antibodies (Jackson Immunosciences), the blots were developed using the chemiluminescence detection kit ECL-Plus (Amersham) according to the manufacturer's instructions. Quantification was performed on scanned images of blots using the Image Lab software, and the values shown on the graphs represent normalization of the protein content evaluated through lamin B1 or tubulin immunoblotting.
Western blotting were performed using standard methods. Blots were incubated with primary antibodies against RECQ1 (Santa Cruz Biotechnology), SMARCAL1 (Bethyl), MRE11 (Novus Biological), DNA2 (Abcam), EXO1 (Santa Cruz Biotechnology), anti-PAR (Abcam), tubulin (Sigma-Aldrich) and lamin B1 (Abcam). After incubations with horseradish peroxidase-linked secondary antibodies (Jackson Immunosciences), the blots were developed using the chemiluminescence detection kit ECL-Plus (Amersham) according to the manufacturer's instructions. Quantification was performed on scanned images of blots using the Image Lab software, and the values shown on the graphs represent normalization of the protein content evaluated through lamin B1 or tubulin immunoblotting.