Ab70469

Briefly, B16F10 cells (~1 × 10⁷) were incubated with 1% formaldehyde for 10 min for crosslink, with adding glycine for a final concentration of 0.125 M to stop crosslink. Then the nuclear pellets were prepared, and suspended with ChIP lysis buffer. The DNA was fragmented with sonication. Immunoprecipitation was performed with antibodies anti-Mi-2β (ab70469, Abcam), anti-Stat1 (ab239360, Abcam) and IgG control at 4 °C for overnight. The complex was pulled down with A/G agarose beads (#20422, Thermo Fisher Scientific) and crosslink was reversed with heating at 65 °C for overnight. The DNA was purified and eluted for quantitative PCR assay. Primers were designed based on the binding peak analysis with ChIP-Atlas-Peak Browser. All data were normalized to gene desert regions of the IgH loci. The real-time PCR was performed in triplicate. Values of [Δ][Δ] Ct method was used to calculate the relative binding enrichment, with the formula: Ct, template (antibody) − Ct, template (IgG) = [Δ] Ct, and the fold enrichments ([Δ][Δ]Ct) were determined using the formula of 2-[Δ] Ct. (experimental)/2-[Δ] Ct (IgH). Standard error from the mean was calculated from replicate [Δ][Δ] Ct values from independent experiments. Primers for ChIP assays are shown in Supplementary data 3.

The purified rabbit polyclonal carboxy terminus-specific anti-Ikaros antibody and the mouse monoclonal anti-ER and anti-Cre antibodies were generated in-house. The N terminus-specific anti-Ikaros (sc-13039, Santa Cruz), anti-Suz12 (3737, Cell Signaling; sc46264, Santa Cruz), anti-Ezh2 (3147S, Cell Signaling), anti-Mta2 (ab8106, Abcam), anti-Mi2β (CHD4; ab70469, Abcam), anti-H3K27me3 (07-449, Millipore), anti-H3K4me3 (ab8580, Abcam), anti-H3 (06-755, Millipore; ab1791 Abcam), anti-H4 (ab7311, Abcam) and anti-β-actin (A5441; Sigma) antibodies were purchased.

Whole WT (Chd4^F/F; Corin^wt/wt) and Chd4 (Chd4^F/F; Corin^cre/cre) mutant hearts were dissected in PBS and nuclear extracts were obtained [9 (link)]. Chd4 was immunoprecipitated with a mouse monoclonal anti-Chd4 antibody (ab70469, Abcam, Cambridge, UK). Proteins were in-gel digested with trypsin at a 5:1 protein: trypsin (w/w) ratio. Peptides were analyzed using LC-MS/MS [34 (link)].
The MS/MS raw files were searched against the Mouse Swissprot database using Sequest running in Proteome Discoverer 1.4. Peptide identification was validated using the probability ratio method [35 (link)] with an additional filtering for precursor mass tolerance of 12 ppm [36 (link)]. The false discovery rate (FDR) was calculated using decoy databases. Peptide and scan-counting were performed assuming as positive events those with an FDR equal to or below 5%.

The lysis buffer (50 mM Tris pH 7.4, 1% Triton X-100, 0.5 mM EDTA, 0.5 mM EGTA, 150 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride and complete protease inhibitor cocktail (Roche)) were used to prepare the whole-cell lysates, which was followed by homogenization and centrifuge (14,000 rpm for 15 min at 4 °C). Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) was used to detect protein concentration. After SDS-PAGE separation and PVDF membrane (BIO-RAD) transfer of the proteins, the specific primary was probed at 4 °C for overnight, before incubated with corresponding horseradish peroxidase (HRP)-conjugated 2nd antibodies. Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) was used for protein detection. Antibodies were: anti-Mi-2β(1:1000), anti-EZH2 (ab70469, Abcam) (1:1000), anti-β-actin-peroxidase antibody (AC15) (1:5000) and anti-rabbit secondary antibody (A-4914) (1:10,000). Antibody information have shown in Supplementary data 3.

Detection of antigens was performed on 5-µm or 10-µm frontal sections through the lung region of paraffin-embedded embryos. Endogenous peroxidases were blocked by incubation in 6% H₂O₂ for 20 min. Antigen retrieval was achieved by citrate-based heat unmasking (H-3300, Vector Laboratories Inc., Burlingame, CA, USA). The following primary antibodies were used: anti-CBX3 (1:200; #PA5-30954, ThermoFisher Scientific, Waltham, MA, USA), anti-CHD4 (1:200; ab70469, Abcam plc, Cambridge, UK), anti-HDAC1 (1:200; #PA1-860, ThermoFisher Scientific), anti-HDAC2 (1:200; #51-5100, ThermoFisher Scientific), anti-HMGB2 (1:200; #ab124670, Abcam plc), anti-PBX1 (1:100; #PA5-82100, ThermoFisher Scientific), anti-TBX2 (1:200 or 1:2000; #07-318, Merck Millipore, Darmstadt, Germany), anti-TBX2 (1:200; #sc-514291X, Santa Cruz Biotechnology Inc.). Primary antibodies were detected by directly labeled fluorescence- or biotin-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA; Dianova, Hamburg, Germany). The signal was amplified using a tyramide signal amplification (TSA) system (NEL702001KT, PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instruction. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, #6335.1, Carl Roth, Karlsruhe, Germany).