Ab8898

Proteins were isolated in RIPA buffer (Pierce, Rockford, IL) on ice. Lysates were separated by electrophoresis on 12% SDS-PAGE polyacrylamide gels. Then, samples were electro-transferred to PVDF membranes (0.22 μm, Millipore) using a wet transfer method and blocked with 5% BSA for 1 h at room temperature. Then, membranes were incubated overnight at 4 °C with primary antibodies of H3 (#4499, CST, 1:2000), H3K9me3 (#ab8898, Abcam, 1:1000) and H3K27me3 (#9733S, CST, 1:1000), H3K4me3 (#9751, CST, 1:1000), H3K9ac (#9649, CST, 1:1000), H3K27ac (#8173, CST, 1:1000), respectively. The next day, blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA) at room temperature for 1 h. Finally, ECL reagent (Millipore) was used for the visualization and detection of antibody-antigen complexes. The uncropped gel images are shown in Supplementary Fig. 8.

The antibodies used for ChIP were: H3 (Abcam ab1791, lot no. GR177884-2), H3K27ac (Abcam ab4729, lot no. GR200563-1), H3K27me3 (Millipore 07–499, lot no. 2475696), H3K36me3 (Abcam ab9050, lot no. GR204353-1), H3K4me3 (Abcam ab8580, lot no. GR190202-1), and H3K9me3 (Abcam ab8898, lot no. GR186864-1), and were validated by the company for specificity. For the H3K4me3 antibody, abcam reports strong binding to H3K4me3 but some cross reactivity with H3K4me2 [103 ]. The following primary antibodies were used for immunofluorescence: mouse anti-Nc82 (1∶20; Developmental Studies Hybridoma Bank) and rat anti-Fru^M (1:200) [18 (link)]. The secondary antibodies used for immunofluorescence were Alexa Fluor goat anti-rat 488 (1:1000), goat anti-mouse 633 (1:500), rabbit anti-GFP 488 conjugate (1:600), and streptavidin 488 conjugate (1:4000) (Thermo Fisher).

Antibodies used were directed against H3 (Abcam ab1791), H4 (Merck Millipore), H3.3 (Merck Millipore 09-838), H3K36me3 (Abcam ab9050), H3K9me3 (Abcam ab8898), H4K20me3 (Abcam ab9053), Flag (Sigma Aldrich), HA (Roche) and Actin (Santa Cruz Biotechnology).

Stomach tissues of mice were collected from untreated and treated with RAN + lime and preserved in 10% formalin. Four mice were selected from each group. Tissue samples were dehydrated, paraffin embedded and sectioned with a microtome. Briefly, after blocking for endogenous peroxidase activity, the sections of stomach tissues were incubated with anti-securin (DCS-280; ab3305; Abcam, UK), anti-H3K4me3 (histone H3 lysine 4 trimethylation) primary antibody (ab8580; Abcam, UK), anti-H3K9me3 (histone H3 lysine 9 trimethylation) primary antibody (ab8898; Abcam, UK), anti-H3K9Ac (histone H3 lysine 9 acetylation) primary antibody (ab12179; Abcam, UK), anti-H3K18ac (H3 lysine 18 acetylation) primary antibody (ab1191; Abcam, USA), anti-Rb-phosphorylation primary antibody (SC-271930; Santa Cruz Biotechnology, USA), anti-E2F1 primary antibody (SC-22820; Santa Cruz Biotechnology, USA) and anti-KAT2A/GCN5 antibody (ab18381; Abcam, USA). IHC analysis was performed with a Strept-Avidin Biotin Kit (Dako, Agilent Technologies Company, Denmark). The scoring of immunohistochemical stains in each specimen was determined using a histological score (H) [33 (link)] (please see Additional file 1). Only Histone 3 antibody (ab1791; Abcam, UK) was used as an internal control.

A total of 8 μg of Clr4 and Clr4 MUT12 were mixed with 5 μg of nucleosome, 215 μM SAM (S-adenosyl Methionine) and 36 mM of Tris pH 8. The samples were incubated at 37°C for the time of the reaction. After each time point, SDS loading buffer was added to the tube and the reaction was inactivated at 95°C for 4 min.
For the western blot, the samples were loaded on a 17% SDS gel ran at 300V and the transfer was done at 15V. The membrane was incubated in a 5% blocking buffer for 2 h against H3K9me1 (ab8896, Abcam), H3K9me2 (ab1220, Abcam), H3K9me3 (ab8898, Abcam) or H3 (ab1791, Abcam), then in the first antibody for 1 h. The membrane was then washed with the 1 × TBST buffer. Then it was incubated with the secondary antibody for 1 h. The membrane was washed with the 1 × TBST buffer then mixed with the chemiluminescence substrate Pico plus for the revelation.

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.