Igg mouse

For immunoprecipitation (IP) assays, cells were lysed in IP buffer with protease-phosphatase inhibitors (20 mM Tris–HCl pH 8, 137 mM NaCl, 1% NP-40 and 2 mM EDTA) at 4°C for 30 min under constant rotation. Lysates were cleared by centrifugation at 12 000 rpm for 20 min at 4°C. Protein concentration was determined and 800–1000 μg of proteins were pre-cleared with 25 μl of Dynabeads G (Invitrogen) for 2 h under constant rotation at 4°C. In parallel, 5 μg of primary antibody and the corresponding control IgG were incubated with 20 μl of Dynabeads G for 2 h under constant rotation at room temperature. Finally, the Dynabeads with the antibodies were washed three times with citrate phosphate pH5 buffer with 0,01% Tween-20 and incubated with the pre-cleared samples overnight at 4°C under constant rotation. The day after, samples were washed three times with PBS (Gibco-Life Technology, Waltham, MA, USA) with proteases and phosphatases inhibitors and boiled in Laemmli buffer at 95°C for 5 min. Pelleted beads were separated using a magnetic separation rack and the supernatants were used for subsequent Western blot analysis. Antibodies used were: SLU7 (BD Bioscience, Franklin Lakes, NJ, USA 612604; 5 μg), DNMT1 (Abcam, ab13537; 5 μg), IgG mouse (Santa Cruz, SC2025; 5 μg).