Rabbit anti ezh2

Paraffin-embedded xenografts or patients’ tissue samples were incubated for 2 h at 56°C for deparaffinization. Antigens were retrieved by microwave treatment in citrate buffer for 15 min to restore antigenicity. After peroxidase activity was blocked with 3% H2O2/methanol for 10 min, sections were incubated with normal goat serum for 20 min to block non-specific antibody binding sites. Sections were incubated with the primary antibodies for 1 h at 25°C and then with biotinylated anti-rabbit/mouse IgG and peroxidase-labeled streptavidin for 10 min each. The following primary antibodies were used: Rabbit anti-EZH2, anti-SMAD2, anti-RB, anti-CDK4 and anti-CCND1 (1:100, Cell Signaling Technology), anti-CCNE1 (1:100, R&D Systems).

Total protein extracts and pulled-down samples were boiled in sample buffer and loaded onto Mini-PROTEAN TGX precast gels (Biorad). Proteins were transferred to a PVDF membrane (Trans-Blot Turbo Transfer System, Biorad), and probed for the following antibodies: Mouse anti-FLAG (1/1000, Sigma F1804), Mouse anti-total H3 (1/1000, Cell Signaling 3638), Rabbit anti-Histone H3.3 (1/1000, Clone RM190 Rev mab biosciences 31-1058-00), Rabbit anti-H3K27me³ (1/1000, Cell signaling 9733), Mouse anti-H4 (1/1000, Active motif 61521), Rabbit anti-H3.3K27M (1/1000, Merck ABE419), Rabbit anti-H3K27me³ (1/1000, Merck 07–449), Rabbit anti-EZH2 (1/1000, Cell signaling 5246), Mouse Anti-GAPDH (1/5000, Fitzgerald 10R-G109A), Goat anti-MCM2 (1/1000, Bethyl A300-122A), Rabbit anti-MCM7 (1/1000, Bethyl A302-584A), and Rabbit anti-MCM4 (1/1000, Bethyl A300-193A). HRP labelled Goat anti-Mouse or Rabbit, or Donkey anti-Goat secondary antibodies were used to visualize protein expression using chemiluminescence substrate (SuperSignal West Dura Extended Duration Substrate, Thermo 34076) on a ChemiDoc system (Biorad). Densitometry analysis was performed with the ImageStudio Lite v5.2.5 (Licor).

Primary antibodies used in this study include rabbit anti-5hmC (Active Motif, #39769), mouse anti-5mC (Active Motif, #39649), rabbit anti-NANOG (Abcam, ab80892), rabbit anti-H3K9me2 (Abcam, ab1220), rabbit anti-H3K9me3 (Abcam, ab8898), mouse anti-H3K27me3 (Wako, MABI0323), rabbit anti-H2AK119ub (Cell signaling, #8240), mouse anti-γH2AX (Sigma-Aldrich, 07–164), mouse anti-SCP3 (Santa Cruz, sc-74569), rabbit anti-SCP3 (Novus, NB-300-232), mouse anti-HP1γ (Invitrogen, MA3-054), human anti-Centromere Protein (CREST) (Antibodies Incorporated, 15–235), rabbit anti-TET1 (Millipore, 09–872), mouse anti-TET1 (Active Motif, 91172), rabbit anti-EZH2 (Cell signaling, #5246), rabbit anti-GFP antibody (Invitrogen, A11122), and rabbit anti-RING1B (Cell signaling, #5694). The secondary antibodies used in this study included Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 568 goat anti-mouse IgG, Alexa Fluor 568 goat anti-rabbit IgG, and Alexa Fluor 647 goat anti-human IgG (Invitrogen).

U87, U251, and D54 cells were fixed with 4% paraformaldehyde solution (PFA) at room temperature for 20 min and washed with PBS. Then, cells were permeabilized and blocked (1% BSA, 0.2% Triton X-100 in PBS) at room temperature for 30 min. Next, cells were incubated for 24 h with rabbit anti-EZH2 (5246S, Cell Signaling, USA) at 4°C and rinsed with PBST (PBS with 0.05% Tween). Later, cells were incubated with Goat anti-Rabbit IgG Alexa Fluor 647 (21246, Santa Cruz Biotechnology, USA) at room temperature for 60 min and rinsed with PBST. Nuclei were stained with 1 mg/mL Hoechst 33342 solution (Thermo Fisher Scientific, USA) at room temperature and again rinsed with PBST. Finally, cells were coverslipped using a fluorescence mounting medium (Polysciences Inc., USA) and visualized in an Olympus Bx43 microscope. For each condition, six arbitrary fields at 400X magnification were captured, and fluorescence density was measured with ImageJ software.

Primary antibodies used were rabbit anti-Ezh2 (Cell Signaling Technology), rabbit anti-H3K27me3 (Cell Signaling Technology), rabbit anti-α Tubulin (Abcam), rabbit anti-H3 (Cell Signaling Technology), mouse anti-Reelin (Millipore), rabbit anti-GRASP65 (Abcam), rabbit anti-Cux1 (Santa Cruze), rabbit normal IgG (Applygen). Second antibodies included anti-rabbit IgG IRDye 680 and anti-mouse IgG IRDye 800 (LICOR Bioscience) for western blot and Alexa 488 anti-rabbit IgG, alexa 555 anti-rabbit IgG (Invitrogen) for immunostaining.

Cells were fixed with 4% paraformaldehyde and sonicated to prepare the chromatin fragments. Chromatin samples were immunoprecipitated with following antibodies at 4°C for 3 h: Rabbit anti-EZH2 (Cell Signaling Technology, Danvers, USA), Rabbit anti-LSD1 (Cell Signaling Technology), Rabbit anti-H3K4eme3 (Abcam, Cambridge, USA), H3K27me3 (Abcam), or normal rabbit IgG (Santa Cruz) antibodies. After crossing reversal, precipitated DNA was analyzed by PCR to detect a 167 bp fragment of miR-663b promoter region (see Supplementary Table S2 for primers sequence). The data were calculated by normalizing against that of corresponding DNA precipitated by rabbit IgG.