Protein lysates of ESCC cells were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22-mm NC membranes (Sigma), and incubated with specific antibodies: anti-CDK6, anti-CDK4, anti-CDK2, anti-cyclinD1, anti-cyclinD3, anti-p27, anti-p21, anti-CDKN2C (Abcam, Shanghai, China), anti-EZH2, anti-EED, anti-SUZ12, anti-EZH1, and anti-β-actin (Cell Signaling Technology). The dilution ratio of the primary antibodies was 1:1,000, although anti-β-actin was diluted to 1:8,000 for Western blotting. Protein bands were visualized with Super Signal Chemiluminescence Substrate (Thermo Scientific) and β-actin was used as a control.
Anti eed
Manufactured by Cell Signaling Technology
Sourced in China
Anti-EED is a primary antibody that specifically recognizes the EED (Embryonic Ectoderm Development) protein. EED is a component of the Polycomb Repressive Complex 2 (PRC2) and plays a role in the epigenetic regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Anti suz12, by Cell Signaling Technology (3 mentions)
Anti ezh2, by Cell Signaling Technology (2 mentions)
Anti h3k27me3, by Cell Signaling Technology (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Anti p21, by Abcam (1 mentions)
0.22 mm nc membrane, by Merck Group (1 mentions)
Anti cyclin d3, by Abcam (1 mentions)
Anti cdkn2c, by Abcam (1 mentions)
Anti ezh1, by Cell Signaling Technology (1 mentions)
Anti cdk4, by Abcam (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Supersignal chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Anti cdk6, by Abcam (1 mentions)
Anti cdk2, by Abcam (1 mentions)
Anti p27, by Abcam (1 mentions)
Anti cyclin d1, by Abcam (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Dapi solution, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
7 protocols using anti eed
1
Protein Expression Analysis in ESCC
2
Western Blot Analysis of EZH2 and EED
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Protein concentrations from RNA pulldown assays were determined with a BCA Protein Assay Kit (cat. no. P0011; Beyotime Institute of Biotechnology). Proteins were denatured for 10 min at 100°C in loading buffer [50 mM Tris-HCl (pH 6.8), 2% SDS (w/v), 0.1% BPB (w/v), 10% glycerol (v/v), 1% β-mercaptoethanol (v/v)], separated by SDS-PAGE (10%), transferred into PVDF membranes and blocked with 5% skim milk for 2 h at room temperature. Membranes were incubated with anti-EZH2 (cat. no. 5246; Cell Signaling Technology, Inc.) or anti-EED (cat. no. 85322; Cell Signaling Technology, Inc.) antibodies, followed by incubation with HRP-conjugated goat anti-rabbit secondary antibody (cat. no. 7074S, Cell Signaling Technology). All antibodies were diluted with 5% skim milk at 1:1,000 and incubated with the membranes for 2 h at room temperature. Protein bands were visualized with the Tanon High-sig ECL Western Blotting Substrate (cat. no. 180-501; Tanon Science and Technology Co., Ltd.).
3
Immunofluorescence Staining of Cryosections and Primary Cells
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Immunofluorescence of cryosections was performed on glass slides. Immunofluorescence for primary cells was performed on glass cover slips. Primary cells were fixed in 4% PFA for 10 minutes at room temperature. Antibody incubations were performed in blocking solution (0.1% Triton X-100, 1% BSA, 10% donkey serum) for 2hr at room temperature or overnight at 4°C. Slides or cover slips were mounted in ProLong Diamond Antifade Mountant (Thermo Scientific, cat# P36965). Primary antibodies used in this study were anti-Sox9 (1:500, Proteintech, cat# 67439-1-Ig), anti-Runx2 (1:500, Cell Signaling, cat# D1L7F), anti-Ki67 (1:2000, Abcam, cat# ab15580), anti-Vimentin (1:1000, Abcam, cat# ab8069), anti-BrdU (1:1000, Abcam, cat# ab6326), anti-Sox10 (1:500, Cell Signaling, cat# D5V9L), anti-ALPL (1:1000, Invitrogen, cat# PA5-47419), anti-Col1a2 (1:500, Proteintech, cat# 66761-1-Ig), anti-Sox6 (1:1000, Proteintech, cat# 14010-1-AP), and anti-Eed (1:1000, Cell Signaling, cat# E4L6E). Secondary antibodies used in this study were Donkey anti-Rabbit IgG (H+L) Alexa Fluor Plus 555 (1:1000, Thermo Scientific, cat# A32794), Donkey anti-Rabbit IgG (H+L) Alexa Fluor 488 (1:1000, Thermo Scientific, cat# A21206), and Donkey anti-Rabbit IgG (H+L) Alexa Fluor Plus 568 (1:1000, Thermo Scientific, cat# A10042). DAPI solution (1:5000, Thermo Scientific, cat# 62248) was added to secondary antibody solutions.
4
Western Blot Analysis of Cellular Proteins
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The primary antibodies used were: anti-GAPDH (sc-47724, Santa Cruz), anti-AR (sc-7305, Santa Cruz), anti-DNMT3A (sc-365769, Santa Cruz), anti-EZH2 (612667, BD Transduction Laboratory), anti-DNMT(5032S, Cell Signaling Technologies), anti-EED (85322S, Cell Signaling Technologies), anti-SUZ12 (3737S, Cell Signaling Technologies), anti-Aurora A (14475T, Cell Signaling Technologies), antiH3K27me3 (9733S, Cell Signaling Technologies), and anti-PLK1 (B290751, BioLegend).
Tumor tissues (25–30 mg) or cellular pellets were lysates with RIPA buffer supplemented with cocktail phosphatase inhibitors (4906845001, Roche) and proteases inhibitors (5892953001, Roche). Protein concentration was determined by BCA reagent (A52255, Thermo Fisher Scientific); 30–50 μg of whole protein lysate was separated on 8–12% SDS–polyacrylamide gels and transferred onto PVDF membrane (88518, Thermo Fisher Scientific). The membranes were blocked with 5% milk in Tris-buffered saline with Tween-20 (TBST) for 30 min at RT, incubated overnight at 4 °C with primary antibodies, and incubated for 1 h at RT with secondary antibodies (anti-rabbit IgG HRP W401B and anti-mouse IgG HRP, W402B, Promega). The protein bands were visualized using the western bright quantum reagent (K-12042-D20, Advansta) and quantified using the Fusion Solo IV LBR system.
Tumor tissues (25–30 mg) or cellular pellets were lysates with RIPA buffer supplemented with cocktail phosphatase inhibitors (4906845001, Roche) and proteases inhibitors (5892953001, Roche). Protein concentration was determined by BCA reagent (A52255, Thermo Fisher Scientific); 30–50 μg of whole protein lysate was separated on 8–12% SDS–polyacrylamide gels and transferred onto PVDF membrane (88518, Thermo Fisher Scientific). The membranes were blocked with 5% milk in Tris-buffered saline with Tween-20 (TBST) for 30 min at RT, incubated overnight at 4 °C with primary antibodies, and incubated for 1 h at RT with secondary antibodies (anti-rabbit IgG HRP W401B and anti-mouse IgG HRP, W402B, Promega). The protein bands were visualized using the western bright quantum reagent (K-12042-D20, Advansta) and quantified using the Fusion Solo IV LBR system.
5
Immunohistochemical Analysis of Brain Tissue
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Animals were deeply anesthetized with Avertin intraperitoneal sodium pentobarbital (Apoteksbolaget AB) and perfused with room temperature phosphate buffer (PB) saline (PBS) through the ascending aorta, followed by ice-cold 4% paraformaldehyde. The brains were subsequently removed, postfixed in the same fixative for 16 to 18 hours, and cryoprotected for 24 to 48 hours in 30% sucrose at 4°C, before being cut on a Leica microtome at a thickness of 30 μm. Sections were permeabilized in 5% bovine serum albumin (BSA) in PBS-Tx100 (PBS with 0.5% Triton X-100), followed by primary antibody incubation at 4°C for 16 to 18 hours using sheep anti-TH (1:1000; catalog no. P60101-150, Pel-Freeze), anti-TPH2 (1:500; catalog no. T0678, Sigma-Aldrich), anti-H3K27me3 (1:500; catalog no. 9733, Cell Signaling Technology), and anti-EED (1:500; catalog no. 85322, Cell Signaling Technology). Fluorescent detection was done with an Alexa-tagged secondary antibody from Molecular Probes, donkey anti-sheep (1:500; catalog no. A21448), goat anti-mouse (1:500; catalog no. A21151), and donkey anti-rabbit (1:500; catalog no. A21206). Section images were obtained in an LSM-700 confocal microscope from Zeiss.
6
CRISPR-Mediated Gene Knockout Validation
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Cells were transfected with the pX459 vector containing sgRNA target site sequences listed in Supplementary Table 1 . Thirty hour after transfection, medium containing 1.5 μg ml−1 puromycin was added, and after a subsequent 48 h, cells were cultured in medium without selection until colonies had grown. Successful homozygous mutation was confirmed by PCR and sequencing using primers in Supplementary Table 1 and, where possible by immunoblotting. Antibodies used were anti-JARID2 (Novus Biologicals NB100-2214) at 1:1,000 dilution, anti-SUZ12 (Cell Signalling 3737) at 1:1,000 dilution, anti-EZH2 (Cell Signalling 5246) at 1:1,000 dilution, anti-ATRX (a gift from R. Gibbons) at 1:10 dilution, anti-EED (a gift of A. Otte) at 1:500 dilution. Controls used were H2AK119u1 (NEB 8240) at 1:1,000 dilution, anti-H3K27me3 (Diagenode pAb-069-050) at 1:1,000 dilution, anti-H3 (Abcam ab1791) at 1:10,000 dilution and anti-RING1B (a gift of H. Koseki) at 1:1,000 dilution.
7
Immunophenotyping of Transcription Factors
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Primary antibodies used were rabbit monoclonal anti-FOXP3 (3 ug/mL, cat. A301-900A; Bethyl Laboratories, Montgomery, TX), rat monoclonal anti-FOXP3 (2 ug/mL, cat. 14-4776-82, lot 4325553; eBioscience), rabbit monoclonal anti-EZH2 (1:1000, cat. 5246, lot 7; Cell Signaling), mouse monoclonal anti-myc (1:1000, cat. 2276, lot 24; Cell Signaling), rabbit monoclonal anti-SUZ12 (1:1000, cat. 3737, lot 6; Cell Signaling), and anti-EED (1:1000, cat. CS204393; Millipore, St. Louis, MO), rabbit monoclonal anti-H3K27me3 (1:1000, cat. CS200603, lot 2819348; Millipore), mouse monoclonal anti-H3 (1:1000, cat. 14269, lot 1; Cell Signaling), mouse monoclonal anti-STAT3 (1:1000, cat. 9139, lot 10; Cell Signaling), and rabbit monoclonal anti-pSTAT3 (1:100, cat. 9131, lot 30; Cell Signaling).
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