Ab139311

ChIP assays were performed using the EZ ChIP kit (Merck Millipore). MPHs or minced liver tissues were fixed in 1% formaldehyde (Sigma-Aldrich) for 10 min. Cross-linking was stopped by addition of glycine at a concentration of 125 mM for 5 min, followed by centrifugation and washing in SDS lysis buffer at room temperature. DNA was sheared to fragments of 200–1,000 bp by sonication. Lysates containing soluble chromatin were incubated and precipitated overnight with 5 µg anti-GR antibody (ab3579; Abcam), anti-TET1 (ab191698; Abcam), anti-TET2 (ab94580; Cell Signaling Technology), anti-TET3 (ab139311; Abcam), or IgG (ab172730; Abcam). DNA–protein immune complexes were removed with protein G agarose and then washed and eluted. Protein–DNA cross-links were reversed by treatment with proteinase K for 2 h at 45°C. The DNA was subsequently purified, diluted, and subjected to qRT-PCR. The mouse Gadd45β gene promoter fragments containing the GRE motif were amplified using the following primers: site 1: 5′-TCT​CAC​TTT​GGG​GAG​AGG​ATC-3′ (forward), 5′-GAG​TCC​CTA​GCC​TTT​CCC​AA-3′ (reverse); site 2: 5′-TTC​GAG​GCC​AGC​CTG​GTC​TA-3′ (forward), 5′-CCT​CAT​GAG​GTC​TCT​GCT​CA-3′ (reverse). The promoter region of the GAPDH proximal promoter was set as the negative control. Primers for the GAPDH promoter were: 5′-CTA​TCC​TGG​GAA​CCA​TCA-3′ (forward) and 5′-AAG​CGT​GTG​GGC​TCC​GAA-3′ (reverse).