Ab12188

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.

At 48 h after transfection with siRNAs, adherent cells were fixed by incubation for 10 min in 4% paraformaldehyde and were then permeabilized by incubation with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 min at room temperature. For blocking, cells were incubated for 1 h at room temperature in PBS containing 7.5% goat serum and 7.5% foetal bovine serum and then incubated overnight at 4°C with the indicated primary antibody. The following antibodies were used for IF: GFP (1/200, ab6673, abcam), PML (1:100, sc966, Santa Cruz), PCAF (1:50, ab12188, Abcam), GCN5 (1:100, 3305, Cell signaling), TRF2 (1:100, IMG-124A, Imgenex), and USP22 (ab4812, Abcam). Secondary labelling was performed using an Alexa Fluor 488- or 594-conjugated antibody (Molecular Probes) at room temperature for 1 h.