Ab8895

The following antibodies were used for western blot and/or ChIP analyses: anti-PU.1/SPI1 (sc325, Santa Cruz Biotechnology, 2258 S, Cell Signaling), anti-KDM1/Lsd1 (ab17721, Abcam), anti-Irf8 (5628 S, Cell signaling), anti-HSP60 (sc13966, Santa Cruz Biotechnology), anti-H3K27ac (ab4729, Abcam), anti-H3K4me1 (ab8895, Abcam), anti-H3K4me2 (ab32356, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-Med1/CRSP1/Trap220 (A300-793A, Bethyl Labs), anti-Brd4 (A301-985A100, Bethyl Labs), goat anti-rabbit IgG (656120, Invitrogen). The concentration that each antibody was used is highlighted in the methods section for ChIP and western blots.

Histone proteins were extracted by acid precipitation. Briefly, cells from a 6-well dish were scraped, washed with PBS and resuspended in extraction buffer (PBS, 0.5% Triton-X-100 (9002-93-1, Sigma-Aldrich), 5 mM sodium butyrate (B5887, Sigma-Aldrich)), and allowed to extract for 20 mins. The extract was centrifuged at 14,000× g for 20 mins and the supernatant was discarded. The pellet was resuspended in 0.2 N HCl and left at 4 °C overnight. The mixture was vortexed, and the pH was neutralized with 1 M Tris-HCl pH 8.0. Western blots were performed using typical laboratory procedures with the antibodies: anti-H3K9me3 (1:1000; ab8898, abcam), anti-H3K4me3 (1:1000; ab8580, abcam), anti-H3K4me1 (1:1000; ab8895, abcam), anti-H3K27me3 (1:1000; 07-449, Millipore), anti-H3ac (1:1000; 06-599, Millipore), anti-H4K12ac (1:1000; ab46983, abcam), anti-H4ac (1:1000; 06-886, Millipore), anti-H3K27ac (1:1000; ab4729, abcam), anti-H2AK119ub (1:1000; 8240, Cell Signaling Technology), anti-H3 (1:1000; ab1971, abcam), anti-OCT4 (1:10,000; SC-8628, Santa Cruz), anti-NANOG (1:1000; A300-397A, Bethyl), and anti-GAPDH (1:10,000; MAB374, Millipore). Uncropped raw images of the Western blot membranes are in Supplementary Figure 8.

All cell lines were cultured in DMEM medium with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (Millipore-Sigma) in a humidified incubator with 5% CO₂ at 37 °C.
Initial experiments were performed on primary MEFs derived from 13.5 days NM1 WT and KO embryos^{46 (link)}. In other experiments were used immortalized MEFs (ATCC® CRL-2752) or cell lines derived from these immortalized cells.
The antibodies against H3K9me3 (ab8898), H3K27ac (ab4729), H3K4me1 (ab8895), H3K4me3 (ab8580), H3K9ac (ab10812), H3 (ab1791), γH2AX (ab2893), p53-K370 (ab183544), PCAF (ab12188), Set1 (ab70378), GAPDH (ab8245), and nonspecific rabbit IgG isotype control (ab37415) were purchased from Abcam (Cambridge, MA, USA). The antibody against NM1 has been previously characterized^{2 (link)}. Alexa Fluor 555 Goat Anti-Rabbit (ab150078) and Alexa Fluor 488 Goat Anti-Mouse (ab150117) were used as secondary antibodies for immunofluorescence, and horseradish peroxidase (HRP)-fused Goat Anti-Rabbit (ab6721) and Rabbit Anti-Mouse (ab6728) secondary antibodies for western blottings were purchased from Abcam. Hoechst 43222 (H1399) and ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; P36931) were purchased from Invitrogen, Waltham, MA, USA. All antibodies have been used according to the manufacturers’ protocols.