Anti h3k27me3 antibody

Embryos were collected at 72 h post-activation (hpa) (i.e., after sperm injection). They were subsequently fixed in 4% (m/v) paraformaldehyde for 30 min and permeabilized with 1% (v/v) Triton X-100 for 50 min. After blocking with 1% (m/v) bovine serum albumin (Sigma, A9418, Saint Louis, MO) in PBS for 1 h, the embryos were incubated with an anti-H3K27me3 antibody (Millipore, Temecula, CA, 1:100) overnight at 4°C, followed by incubation with Alexa Fluor 488 mouse anti-rabbit IgG (Invitrogen, Carlsbad, CA, 1:100) for 1 h. After the nuclei were stained with 10 μg/mL Hoechst 33342, the embryos were mounted on slides with DABCO (1,4-diazabicyclo-(2.2.2) octane; Beyotime, P0126, Beijing, China) and observed with a laser-scanning confocal microscope (Zeiss, LSM700, Jena, Germany).

The primary rat HSCs were administrated with EZH2 inhibitor DZNep, DMSO, or were infected with adenovirus recombined with mouse Jmjd3 gene (AD-Jmjd3) or AD-GFP as control. After four days treatment, these cells were harvested and sonicated by bioruptor (Diagenode, Belgium). By using chromatin immunoprecipitation (ChIP) assay kit (Upstate, cat no. 17-371), the supernatant of sonicated cells was co-immunoprecipitated with the anti-H3K27me3 antibody (Millipore, Germany, 07-449) or the anti-H3K27me2/me3 antibody (Active Motif, China, 39535) to assess the binding of EZH2 or JMJD3 respectively, and the mouse IgG was used as negative control. The enrichment of target gene fragment in DNA precipitate was analyzed by quantitative real-time PCR, and the primers for target genes were listed in Table S4. The fold enrichments of target genes between ChIP-DNA and input-DNA were determined by ΔCt.

IF was performed as described previously (Tatavosian et al., 2015 (link); Zhen et al., 2014 (link)). Wild-type (PGK12.1), Eed^−/−, Ezh2^−/−, Y-Eed/Eed^−/−, and Y-Ezh2/Ezh2^−/− mES cells were cultured on coverslips and fixed using 2.0% paraformaldehyde. After permeabilizing with 0.2% Triton X-100, the cells were washed with basic blocking buffer (10 mM PBS pH 7.2, 0.1% Triton X-100, and 0.05% Tween 20) and then blocked with blocking buffer (the basic blocking buffer plus 3% goat serum and 3% bovine serum albumin). Anti-H3K27me3 antibody (07–449; Millipore, Billerica, MA) was incubated with the cells for 2 hr at room temperature. After washing with the basic blocking buffer, Alexa Fluor 488-labelled goat anti–rabbit antibody (A-11008; Life Technologies, Carlsbad, CA) was incubated with the cells for 1 hr. After incubating with 0.1 μg/ml hoechst, the cells were washed and then mounted on slides with ProLong Antifade reagents (P7481; Life Technologies, Carlsbad, CA). The images were taken and processed as described previously (Tatavosian et al., 2015 (link); Zhen et al., 2014 (link)).

Cultured MEFs were washed with cold PBS buffer and then lysed with KALB lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 % (v/v) Triton X-100, 1 mM EDTA pH 7.5) supplemented with 1 mM sodium vanadate, 1 mM PMSF and protease inhibitors (complete mixture tablets; Roche) on ice for 30 min. Insoluble material was removed by centrifugation at 15,000 g at 4°C for 5 min. Total protein concentration in the whole cell extract was quantified using the BCA protein assay kit (Pierce) following manufacturer’s instructions. Proteins were resolved by 4–12 % SDS-PAGE (Invitrogen), transferred to PVDF membranes (Osmonics; GE) and blocked with 5 % (w/v) skim milk powder in 0.1 % (v/v) Tween-PBS for 1 h at room temperature. Membrane was incubated overnight with anti-Setdb1 antibody (1:2000 diluted; Santa Cruz, sc-66884) and anti-Actin antibody (1:2000 diluted; Santa Cruz, sc-1616), anti-H3K27me3 antibody (1:2500; Millipore 07-449), anti-H3K9me2 antibody (a:1000; Abcam, ab1220), anti-H3K9me3 (1:1000; Millipore, 07-442) at 4 °C followed with horseradish peroxidase (HRP)-conjugated secondary antibodies. Membrane was visualised using ECL system (Immobilon; Millipore) following manufacturer’s instructions.

Tobacco leaves (0.8–1.0 g fresh weight) were harvested and used for the H3K27me3 ChIP assay. ChIP was performed with EpiQuik^TM Plant ChIP Kit (Epigentek, http://www.epigentek.com) following the manufacturer’s instructions. The anti-H3K27me3 antibody (Millipore, http://www.emdmillipore.com) was used for immunoprecipitation. 2.0 μL of immunoprecipitated DNA was used for qRT-PCR experiments. Primers used for the qRT-PCR detection are listed in the S2 Table. qRT-PCR was performed with 1×iQ^™ SYBR Green Supermix (Bio-Rad) on CFX96TM Real-Time System (Bio-Rad). The thermal cycling consisted of an initial denaturation (94°C, 3 min) followed by 40 cycles (94°C, 20 s; 56°C, 20 s; 72°C, 30 s). ChIP assays were performed with three biological replicates.