MDA-MB-231 cells seeded in 6 well plates at a cell density of 3 x 105 were treated with HKMTI-1-005 (1–7.5uM) for 48 h. Following lysis in Triton Extraction Buffer (TEB, PBS containing 0.5 % Triton ×100 (v/v), 1/1000 protease inhibitor), nuclei were re-suspended in 0.2 N HCL at a density of 4 x 107 nuclei per millilitre and incubated overnight at 4 °C to acid-extract the histones, before being centrifuged at 6500g for 10 min at 4 °C. Protein concentration was determined using the Bradford assay. H3K27me3, H3K9me3, H3K9me2, H3K9me and total H3 protein expression levels in the histone extract samples were determined using Western blot analysis using H3K27me3 (1:1000; Abcam), H3K9me3 (1:1000; Abcam), H3K9me (1:1000) and H3 (1:2000; Abcam) antibodies. After washing, the membrane was incubated with a horseradish peroxidase-labelled secondary antibody (1 h, room temperature). The membrane was incubated for 1 min with 5 mL of Pierce ECL Western blotting substrate (Thermo Scientific). Images were captured using Konica Minolta SRX101A Tabletop X-Ray film processor.
H3k27me3
Manufactured by Abcam
Sourced in United States, United Kingdom
H3K27me3 is a histone modification that is associated with transcriptional repression. It is a post-translational modification of the histone H3 protein, where the lysine residue at position 27 is trimethylated. This modification is commonly used as a marker for epigenetic regulation and chromatin structure.
Automatically generated - may contain errors
Lab products found in correlation
H3k4me3, by Abcam (33 mentions)
H3k27ac, by Abcam (24 mentions)
H3k9me3, by Abcam (16 mentions)
H3k9me2, by Abcam (8 mentions)
H3k36me3, by Abcam (6 mentions)
H3k4me1, by Abcam (6 mentions)
H3k9ac, by Abcam (6 mentions)
H3k9me3, by Merck Group (6 mentions)
H3k4me3, by Merck Group (6 mentions)
Flag tag (flag), by Merck Group (6 mentions)
Suz12, by Abcam (5 mentions)
β actin, by Santa Cruz Biotechnology (5 mentions)
β actin, by Merck Group (5 mentions)
Ez chip kit, by Merck Group (5 mentions)
Hiseq 2500, by Illumina (4 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Ab6002, by Abcam (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Histone h3, by Abcam (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
140 protocols using h3k27me3
1
Histone Methylation Analysis in TNBC Cells
2
Immunofluorescence Labeling of Chromosomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow-sorted chromosomes (105) were cytospun (Cytospin3, Shandon) on to poly(l -lysine) slides at 1,200 r.p.m. for 10 min, as previously described36 (link). Samples were incubated with blocking buffer (5% normal goat serum in 10 mM Hepes, 2 mM MgCl2, 100 mM KCl and 5 mM EGTA) for 30 min at RT, and incubated overnight at 4 °C with primary antibodies to Cenpa (catalog no. 2048S, Cell Signaling), H3K9me3 (catalog no. 07-523 or 07-442, Millipore), H3K27me3 (catalog no. ab6002, Abcam or catalog no. 07-449, Millipore), Esrrb (catalog no. PP-H6705-00, Perseus Proteomics) and Sox2 (catalog no. ab97959, Abcam). All antibodies were diluted 1:200 in blocking buffer. Chromosomes were incubated with appropriate secondary antibodies (anti-mouse Alexa 488 (catalog no. A11001, Invitrogen), anti-rabbit Alexa 488 (catalog no. A11008, Invitrogen) or anti-mouse Alexa 568 (catalog no. A11031, Invitrogen)) for 1 h at RT. All secondary antibodies were diluted 1:400 in blocking buffer. Stained chromosomes were mounted in DAPI-containing Vectashield (Vector Laboratories). Wide-field epifluorescence microscopy was performed on an Olympus IX70 inverted microscope using a UPlanApo ×100/1.35 oil objective lens and Micro-Manager software.
3
Comprehensive Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed using the cell lysate, according to standard protocols. Briefly, proteins were separated by SDS-PAGE and transferred to nitrocellulose filter membranes; subjected to immunoblotting with antibodies. The following antibodies were used: α-tubulin (Cell Signaling Technology, Cat # 3873S, RRID:AB_1904178, 1:5000), ACLY (Abcam, Cat # ab40793, RRID:AB_722533, 1:1000), SCD1 (Abcam, Cat # ab236868, 1:1000), FASN (Cell Signaling Technology, Cat # 3180S, RRID:AB_2100796, 1:1000), ELOVL2 (Abcam, Cat #ab176327, 1:1000), H3 (Proteintech, Cat # 17168-1, RRID:AB_2716755, 1:1000), H3K27me3 (Abcam, Cat # ab6002, RRID:AB_305237, 1:1000), β-ACTIN (Proteintech, Cat # 66009-1, RRID:AB_2687938, 1:2000), EZH2(CST, Cat # 5246S, RRID:AB_10694683, 1:2000). Secondary antibodies conjugated to LI-COR IRDye were obtained from LICOR Biosciences (Lincoln, Cat # 925-68071, RRID:AB_2721181, Cat # 925-68072, RRID:AB_2814912, 1:10000). The membranes were visualized using Odyssey Imager (LICOR Biosciences). All antibodies were validated by the commercial vendor.
4
Profiling Chromatin Modifications in BT12 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BT12 cells were incubated with 100 nM mithramycin or PBS control for 8 or 18 h. Cells were cross‐linked, lysed, and sheared as described (Harlow et al, 2019 ). 10 µg solubilized chromatin was immunoprecipitated with 1 µg mouse IgG and 1 µg H3K27me3 (Abcam); 2 µg rabbit IgG and 2 µg SMARCC1 (Cell Signaling); 1 µg rabbit IgG; and 1 µg H3K27ac (Active Motif). Antibody–chromatin complexes were immunoprecipitated and purified as described (Harlow et al, 2019 ). ChIP DNA was quantified with SYBR green relative to a standard curve generated with chromatin from the respective sample for each primer set. qPCR as described above was performed with the following primer sets (GAPDH, MYT1, SOX2, CCND1, SP1). Antibody information and PCR primer sequences are available in Appendix Table S5 .
5
Sorafenib and EPZ-6438 Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DMEM/F12K, trypsin, penicillin and streptomycin were purchased from Life technology. Sorafenib was purchased from Sigma (St. Louis, MO, USA). The EZH2 inhibitor EPZ‐6438 was obtained from Adooq Bioscience (Shanghai, China). Antibodies against RAF and VEGFP were brought from Abcam (Cambridge, MA, USA). The anti‐GAPDH and anti‐H3 antibodies were obtained from Sigma. Antibodies used to detect the protein levels of EZH2, H3K27me3 and H3K27Ac were purchased from Abcam. The secondary antibodies for Western blot were purchased from Jackson ImmunoReseach (West Grove, PA).
6
ChIP-seq analysis of chromatin modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP was performed for SUZ12 (Abcam), EZH2 (Cell Signaling), JARID2 (Cell Signalling), H3K27me3 (Abcam), total H3 (Abcam), and EP300 (Bethyl) as previously described (Kanhere et al. 2012 (link)), with variations described in the Supplemental Methods . Enrichment of specific gene sequences was measured by qPCR (Applied Biosystems). Libraries were sequenced using an Illumina HiSeq 2500 (50 bp single-end). ChIP-seq reads were trimmed to remove adaptors and low-quality bases and aligned with Bowtie 2 (Langmead and Salzberg 2012 (link)). MACS14 (Zhang et al. 2008 (link)) and MAnorm (Shao et al. 2012 (link)) were used to identify sites at which SUZ12 binding was significantly changed by RNase treatment (P < 0.05).
7
Western Blot Analysis of Chromatin Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with NP-40 buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM ethylenediaminetetraacetic acid (EDTA)). Antibodies against actin (Zhongshan Golden Bridge Biotechnology), EZH2 (Cell Signaling Technology #9441), TFIIIC102 (Santa Cruz sc-101176), TFIIIC110 (Santa Cruz sc-81406), TFIIIC63 (Santa Cruz sc-134082), SUZ12 (CST #3737), H3 (CST #9715), H3K27me3 (Abcam ab6002) were purchased from commercial sources.
8
Quantitative Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular extracts were prepared using a lysis buffer containing 20 mM Tris HCl (pH 7.5), 150 mM NaCl, 15% glycerol, 1% Triton, 25mM β-glycerolphosphate, 50mM NaF, 10mM NaPyrophosphate, orthovanadate, PMSF (all chemicals are from Sigma-Aldrich, St. Louis, MO), Protease inhibitor cocktail (Roche) and 2% SDS. 25 U of Benzonase® Nuclease (EMD Chemicals, Gibbstown, NJ) and 20mM of DTT (Sigma) were added to the lysis buffer right before use. 15 µg of protein (determied by Bradford) was loaded, separated by 4–20% SDS-PAGE, and transferred to polyvinylidene difluoride membranes, blocked with 5% nonfat dry milk for 60 minutes at rt, and incubated overnight at 4°C with primary antibody. After incubation for one hour with horseradish peroxidase-conjugated secondary antibodies, they were visualized by enhanced chemiluminescence (Millipore Corp, Billerica, MA). Antibodies used in this study are: H3K27me3 (1/1000, Abcam, ab6002), H3K9me3 (1/1000, Abcam, ab8898), H3K9/14Ac (1/1000, Cell Signaling, 9677s), EED (1/1000, gift from Dr. Bomsztyk66 (link)), HIF1α (1/2000, BD Biosciences, 610958), LDHA (1/1000, Cell Signaling, 3582), JARID2 (1/1000, Cell Signaling, 13594), pSTAT3 (1/1000, Abcam, Ab76315) and γ-tubulin (1/10000, Promega, G712A).
9
Chromatin Immunoprecipitation and Sequencing
10
Quantitative Analysis of BAP1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For quantitative PCR, a standard protocol was followed. Total RNA was extracted with Trizol and was converted to cDNA. Primers for BAP1 were 5'-GCT CGT GGA AGA TTT CGG TGT-3' as forward, and 5'- TCA TCA ATC ACG GAC GTA TCA TC-3' as reverse. GAPDH was used as internal reference. cDNA was subject to the ABI 7500 for quantitative PCR procedure and the program was per manufacturer's instruction of SYBR Green system. The expression of BAP1 was calculated according to internal references and expressed as folds over the control group.
For western blotting, total protein of cell lysates was extracted and equal protein amount of 25 μg was loaded onto 10% sodium dodecyl sulphate polyacrylamide gel. Gels were subsequently transferred to nitrocellulose membrane. The membranes were blockedd with 5% non-fat milk. Primary anti-human BAP1 (Cell Signaling), EZH2 (Abcam), H3K27me3 (Abcam), and Actin (Abcam) antibodies were then added at the dose recommended by the manufacturers and membranes were kept at 4°C overnight. Procedure was finalized by enhanced chemiluminescence.
For western blotting, total protein of cell lysates was extracted and equal protein amount of 25 μg was loaded onto 10% sodium dodecyl sulphate polyacrylamide gel. Gels were subsequently transferred to nitrocellulose membrane. The membranes were blockedd with 5% non-fat milk. Primary anti-human BAP1 (Cell Signaling), EZH2 (Abcam), H3K27me3 (Abcam), and Actin (Abcam) antibodies were then added at the dose recommended by the manufacturers and membranes were kept at 4°C overnight. Procedure was finalized by enhanced chemiluminescence.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!