Anti dnmt3a

Mice were perfused with 4% PFA after deep isoflurane before being analyzed by immunohistochemistry. The DRGs were collected and post-fixed in 4% PFA overnight, followed by dehydrating in 30% sucrose for two nights at 4°C. Finally, the DRGs were sectioned at 15–20 μm and kept them in −80°C refrigerator.
Before primary antibody incubation, the section was blocked in 1X PBS with 10% donkey serum and 0.3% Triton X-100. The sections were then incubated with anti-DNMT3a (Santa Cruz, Dallas, TX, USA) overnight at 4°C followed by incubating secondary antibody conjugated to Cy3 (1:500, Jackson ImmunoResearch, West Grove, PA, USA) for 2 h. Finally, the sections were mounted using Fluoroshield™ with DAPI (Cat. No: F6057, Sigma-Aldrich, Burlington, MA, USA).

Chromatin immunoprecipitation (ChIP) assays were performed essentially as described before [67 (link), 68 (link)]. Briefly, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mMTris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ~200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-MRTF-A (Santa Cruz, sc-10768), anti-DNMT1 (Santa Cruz, sc-20701), anti-DNMT3a (Santa Cruz, sc-20703), or anti-DNMT3b (Santa Cruz, sc-20704). Precipitated genomic DNA was amplified by real-time PCR with the following primers: ICAM-1 proximal promoter, 5'-CCCTGCCACCGCCGCC-3' and 5'-AGGGGCGGTGCTGCTTTCC-3'; ICAM-1 intronic region, 5'-AATTCCAGAGCTGACTTATCC-3' and 5'-ATCTCAGGCTTTGTTGAGC-3'; SIRT6 promoter, 5'-AACTCTGCGTGGCATTCAAA-3' and 5'-AAATGCGGGACACAGGCTAT-3'. A total of 10% of the starting material is also included as the input. Data are then normalized to the input and expressed as % recovery relative to the input as previously described [56 (link), 69 (link)]. All experiments were performed in triplicate wells and repeated three times.

After a 16 h doxycycline (2 μg/mL) induction, whole-cell extracts were obtained from 5 × 10⁶ Bio-Dnmt3a1 ES cells by resuspension in CytoBuster Protein Extraction Reagent (Millipore) followed by 15-min incubation at room temperature (RT). Non-induced ESCs served as a negative control. Samples were spun at 20,000 g, 4 °C for 15 min and supernatant was isolated. Immunoprecipitation of biotinylated-DNMT3A1 was performed with M280-streptavidin beads (Thermo Fisher) using whole-cell extract from cultures with or without doxycycline induction. Equal amounts of protein were resuspended in Laemmli buffer and loaded for western blot analysis. The following antibodies were used: anti-DNMT3A (Santa Cruz, sc-20,703), anti-TET1 (provided by Dr. Guoliang Xu, [24 (link)]), and anti-β-actin (Santa Cruz, sc-47,778).