ChIP assays were conducted using the ChIP Assay Kit (Beyotime, Shanghai, China) as reported (8). Briefly, 1 × 106 WT BMDMs were cross-linked with formaldehyde, and chromatin fragmentation was carried out according to the protocol provided. The above-prepared diluted soluble chromatin solution was then incubated with an Mbd2 antibody overnight at 4°C with rotation. Normal rabbit immunoglobulin G was used to determine nonspecific bindings. The above mixtures were next incubated with protein A + G agarose beads, and the protein-DNA complexes were eluted out after washes. The eluted DNA was subjected to ChIP-PCR with indicated primers. The primers used for Ship in the ChIP assay were as follows: F1, 5′-TCA GAA TGT AGA CGT GAG CTC TTG T-3′; R1, 5′-GCT GTG TTG ATG TCA TCC ATG G-3′; F2, 5′-TCC CAT GTC CTC CAC ACA CCA G-3′; R2, 5′-CAC TCC TTC ATC TGA ACC TTG TCA C-3′; F3, 5′-AAC AAT CAC CAC TTC TGC CGT AAG C-3′; R3, 5′-CCT CTG TGG CTA GAC ATG GCT AT-3′; F4, 5′-GGA AGG TAG ATG ATG CCC CC-3′; R4, 5′-CAG CAA AGT AGT TCA GGG CC-3′; F5, 5′-GAG TGT CCG TCC TGG GAG TGG-3′; and R5, 5′-AGT TGG GAG GAG ACG GGT ACT CAC A-3′. The distal region of the Ship promoter absent of CpG DNA was used as a negative control. The primers were F (5′-CAT GAG ATC CAA CTG TAA GGC-3′) and R (5′-CAT ACT GCT GTT CAT CAC CAA-3′). The Dual-Luciferase Reporter System (Promega, Madison, WI) was used for the Ship promoter luciferase reporter assays, in which the Mbd2 binding site within the Ship promoter was disrupted using the established techniques (17).

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