Rabbit anti dnmt1 ab a 1700

B cells were lysed in Laemmli buffer. Cell extracts containing equal amounts of protein (20 μg) were fractionated through 10% SDS–polyacrylamide gel electrophoresis. The fractionated proteins were transferred onto polyvinylidene difluoride membranes (Bio-Rad) overnight (30 V/90 mA) at 4°C. After blocking and overnight incubation at 4°C with mouse anti-AID mAb (clone ZA001; Invitrogen), mouse anti–NF-κB p65 mAb (clone D14E12; Cell Signaling Technology), rabbit anti–Acetyl-(Lys310)NF-κB p65 mAb (clone D2S3J; Cell Signaling Technology), rabbit anti–Acetyl-DNMT1-K1127/K1129/K1131/K1133 Ab (A5595, ABclonal), rabbit anti-Sirt1 Ab (A11267, ABclonal), rabbit anti-Dnmt1 Ab (A-1700, Epigentek), or rat anti–β-actin mAb (clone AC-15, Sigma), the membranes were incubated with HRP-conjugated secondary Abs. After washing with TBS–Tween 20 (0.05%), bound HRP-conjugated mAbs or Abs were revealed using Western Lightning Plus-ECL reagents (PerkinElmer Life and Analytical Sciences). Densitometry was performed with Fiji software. All the Abs and mAbs used in the above experiments are listed in table S1.

ChIP assays were performed as previously described (6 (link)). B cells (1.0 × 10⁷) were treated with formaldehyde (1.0%, v/v) for 10 min at 25°C to cross-link chromatin. After quenching with 100 mM glycine (pH 8.0) and washing with cold PBS containing protease inhibitors (Roche), B cells were resuspended in lysis buffer [20 mM tris–HCl, 200 mM NaCl, 2 mM EDTA, 0.1% (w/v) SDS, and protease inhibitors (pH 8.0)]. Chromatin was sonicated to yield DNA fragments (about 200 to 600 bp), precleared with Pierce Protein A beads (Thermo Fisher Scientific), and incubated with rabbit anti–NF-κB p65, anti–Acetyl-(Lys310) NF-κB p65 and anti-Sirt1 Abs (as above), rabbit anti-H3K9Ac/K14Ac (17-615, Millipore), rabbit anti-DNMT1 Ab (A-1700, EpiGentek), rabbit anti-Sirt1 Ab (A11267, ABclonal), mouse anti-Dnmt3l mAb (S117-9, StressMarq Biosciences), or control rabbit or mouse IgG with irrelevant specificities at 4°C overnight. Immune complexes were precipitated by Protein A agarose beads, washed, and eluted [50 mM tris-HCl, 0.5% SDS, 200 mM NaCl, and proteinase K (100 μg/ml) (pH 8.0)], followed by incubation at 65°C for 4 hours. DNA was purified using a QIAquick PCR purification kit (Qiagen). The precipitated DNA was used as a template for qPCR analysis involving specific primers (table S1B).