Ab9053

Histone protein samples were separated by SDS-PAGE (12 % gel). After electrophoresis (100 V for 20 min, and 200 V for 30 min), proteins were transferred to a PVDF membrane (180 mA for 60 min). Membrane was incubated with 5 % skimmed milk in TBS-T at room temperature for 1 h, followed by primary antibodies either 1:10,000 H4K20me3 (ab9053, Abcam) or 1:20,000 H3K9me3 (ab8898, Abcam) or 1:20,000 H3K4me3 (ab8580, Abcam) antibodies at 4 °C overnight. After washing, membrane was incubated with 1:10,000 corresponding secondary antibodies for 1 h. The immunocomplex signal was developed using the SuperSignal™ West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) and imaged using the ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories). Histone H4 and Histone H3 were detected as loading controls using 1:10,000 anti-Histone H4 mouse antibodies (mAbcam 31830, Abcam) and 1:20,000 anti-Histone H3 mouse antibodies (14269, Cell Signaling Technology), respectively. Experiments were done in triplicate.

ChIP assay was performed as previously described [15 (link)] with the following antibodies: 3 ug of anti-H3K9me3 (ab8898, abcam), 3 ug of anti-H4K20me3 (ab9053, abcam), 2 ug of anti-TRF2 (NB110–57130, Novus Biologicals), 5 ug of anti-HP-1ɣ (05–690, Sigma) and IgG (sc-2025, Santa Cruz Biotechnology). Samples were transferred to a Hybond-N⁺ membrane (Amersham) on a dot blot, and hybridized with the same telomeric probe (TTAGGG)n used for TRF. The signal was quantified with the ImageJ software. The amount of telomeric DNA in each ChIP was normalized to total telomeric DNA signal.

Embryos were fixed in 4% paraformaldehyde in PBS at room temperature (RT) for 20 min after the removal of the zona pellucidae with acid Tyrode’s solution (pH
2.5). After washing three times in PBS containing 0.3% polyvinylpyrrolidone (PVP K-30; Nacalai Tesque), fixed embryos were treated with 0.5% Triton X-100
(Sigma-Aldrich) in PBS at RT for 40 min, and were blocked in PBST containing 1.5% BSA, 0.2% sodium azide, and 0.02% Tween20 (antibody dilution buffer) at RT for
1 h. Embryos were incubated with primary antibody in antibody dilution buffer at 4°C overnight, for H4K20me1 (1:3000 dilution; ab9051, Abcam, Cambridge, UK),
H4K20me3 (1:5000 dilution; ab9053, Abcam), γH2AX (1:200 dilution; AB_315794, Biolegend, San Diego, CA, USA), and FLAG (1:5000 dilution; F1804, Sigma-Aldrich)
staining. Embryos were washed three times in antibody dilution buffer, and then were incubated with appropriate secondary antibody in antibody dilution buffer
(1:500 dilution; Alexa Fluor 488-conjugated goat anti-mouse IgG or Alexa Fluor 594-conjugated goat anti-rabbit IgG, Invitrogen) at RT for 1 h. After stained
with Hoechst 33342 (Sigma-Aldrich), embryos were mounted on slides in 50% glycerol in PBS and signals were observed using a fluorescence microscopy (BX50 or
FSX100, Olympus, Tokyo, Japan). The number of analyzed embryos is given in each figure legend.