Ab51023

Co-IP assays were performed using M-CSF macrophages differentiated from CD14+ monocytes for 5 days. Cell extracts were prepared in lysis buffer [50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1% Triton-X-100, protease inhibitor cocktail (cOmplete™, Merck)] with corresponding units of Benzonase (Sigma) and incubated at 4°C for 4 h. 50 μl of supernatant was saved as input and diluted with 2× Laemmli sample buffer (4% SDS, 20% glycerol, 120 mM Tris–HCl, pH 6.8). Supernatant was first incubated with PureProteome™ Protein A/G Mix Magnetic Beads (Millipore) for 1 h to remove background signal. Samples then incubated overnight at 4°C with corresponding antibodies against DNMT1 (NB100-56519, Novus), DNMT3A (3598, Cell Signaling), DNMT3B (sc-20704, Santa Cruz), SIRT1 (07-131, Millipore), and SIRT2 (ab51023, Abcam) according to the specifications of each antibody. Negative controls were incubated with rabbit (12-370; Merck Millipore) and mouse (12-371; Merck Millipore) IgGs. Subsequently, samples were incubated with magnetic beads at 4°C for 2 h, and beads were then washed three times with lysis buffer. For sample elution, 100 μl of 1× Laemmli sample buffer was added to beads. Samples and inputs were denatured at 95°C in the presence of 1% β-mercaptoethanol. Whole-cell extracts and IP samples were visualized by western blotting, as described above.

ChIP assays were performed as previously described (16 (link)). Briefly, MOs, DMSO- and cambinol-treated day 5 and LPS-stimulated MACs were crosslinked with 1% methanol-free formaldehyde (Thermo Fisher) for 15 min and subjected to immunoprecipitation after sonication. ChIP experiments were performed using the LowCell# ChIP kit™ protein A (Diagenode, Liège, Belgium). We used antibodies against acetylated lysine 16 of histone H4 (H4K16ac; 07-329, Millipore), monomethylated lysine 4 of histone H3 (H3K4me1; ab8895, Abcam), trimethylated lysine 4 of histone H3 (H3K4me3; CS200580, Millipore), acetylated lysine 27 of histone H3 (H3K27ac; 07-360 Millipore), SIRT1 (07-131, Millipore), SIRT2 (ab51023, Abcam), PU.1 (PA5-17505, Invitrogen) and DNMT3B (sc-20704, Santa Cruz). Corresponding rabbit IgG (Diagenode) is used as control. Protein binding was analyzed by real-time quantitative PCR, and data are represented as ratio of the enriched fraction with respect to input. ChIP primers were designed for the areas flanking differentially methylated CpGs and their sequences are shown in Supplementary Table S1.