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.
H3k9me3
Manufactured by Abcam
Sourced in United States, United Kingdom
H3K9me3 is a histone modification marker that indicates trimethylation of lysine 9 on histone H3. It is commonly used in epigenetic research to study chromatin structure and gene regulation.
Automatically generated - may contain errors
Lab products found in correlation
H3k27me3, by Merck Group (30 mentions)
H3k4me3, by Abcam (24 mentions)
H3k4me3, by Merck Group (19 mentions)
H3k27ac, by Abcam (16 mentions)
H3k27me3, by Abcam (16 mentions)
H3k9me2, by Abcam (15 mentions)
H3k27me3, by Cell Signaling Technology (15 mentions)
H3k36me3, by Abcam (14 mentions)
H3k4me1, by Abcam (11 mentions)
Flag tag (flag), by Merck Group (11 mentions)
β actin, by Merck Group (8 mentions)
H3k9ac, by Merck Group (8 mentions)
H3k4me2, by Merck Group (6 mentions)
H3k27me3, by Active Motif (6 mentions)
H4k20me3, by Abcam (6 mentions)
Histone h3, by Cell Signaling Technology (6 mentions)
H3k27me3, by Diagenode (6 mentions)
H3k4me2, by Abcam (6 mentions)
H3k4me3, by Active Motif (6 mentions)
H3k9me2, by Merck Group (6 mentions)
165 protocols using h3k9me3
1
Histone Methylation Analysis in TNBC Cells
2
ChIP-seq Antibody Validation and Immunofluorescence
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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-FruM (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).
3
Epigenetic and Extracellular Matrix Profiling
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Immunohistochemistry was performed for histone modifications (H3K4me3; Millipore, 07–473; H3K9me3, Abcam, ab8898; H3K27me3, Millipore, 07–449), DNA modifications (5-hydroxymethylcytosine and 5-methylcytosine) and collagen markers (collagen-II and collagen-X). Details on antibodies and precedures are shown in Supplementary Table 1 .
4
Chromatin Immunoprecipitation Protocol
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Chromatin isolation and immunoprecipitation were performed as described previously [21 (link)]. The following antibodies were used for immunoprecipitation: H4K9/14Ac (005–044) from Diagenode; H3K4m2 (32356) and H3K9me3 (8898) from Abcam; H3 (07–690), H3K4me2 (07–030), H3K9me2 (17–681) and H3K27me3 (07–449) from Millipore. Peptide competition assays were performed for H3K4me2, H3K9me2 and H3K27me3 as described previously [21 (link)].
5
Investigating Epigenetic Regulators in A549 Lung Cancer
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A549 human lung cancer cell line was purchased from the Korean Cell Bank. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, and maintained in a humidified incubator at 37℃/5% CO2. The anticancer drugs, cycloheximide (CHX), actinomycin D, and kinase inhibitors were obtained from Sigma Aldrich (Seoul, Korea). The drugs were dissolved in appropriate solvents according to the manufacturer’s protocol. Antibodies for this experiment were purchased from the following companies: SETDB1 (Abcam, MA, USA), SUV39H1 (Upstate Biotechnology, NY, USA), EZH2 (Abcam, MA, USA), p53 (Santa Cruz Biotechnology, CA, USA), FosB (Santa Cruz Biotechnology, CA, USA), H3K9me3 (Abcam, MA, USA), and β-actin (Sigma-Aldrich).
6
Chromatin Profiling of Mouse Hippocampus
7
Quantitative Protein Expression Analysis
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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).
8
Autophagy Induction and Analysis
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Rapamycin was purchased from Millipore. H2O2 was from Fisher Scientific. 4-hydroxytamoxifen and etoposide was from Sigma-Aldrich. The following antibodies are used: LC3 (MBL #PM036 for WB of MEFs; Cell Signaling Technology #3868 for IP, ChIP, IF, WB; Cell Signaling Technology #2775 for WB), β-tubulin (Sigma-Aldrich #T4026), calreticulin (Cell Signaling Technology #12238), COX IV (Cell Signaling Technology #4850), Atg5 (Cell Signaling Technology #8540), Atg7 (Cell Signaling Technology #8558), Lamin B1 (Abcam #ab16048), Lamin B2 (Abcam #ab8983), Lamins A/C (Millipore #MAB3211), GFP (Roche #11 814 460 001 and Abcam #ab290), p62 (Abnova #H00008878-M01), GAPDH (Fitzgerald Industries #10R-G109A), p16 (Abcam # ab16123), Ras (Millipore #05-516), HA (Sigma-Aldrich #H3663), H3K27me3 (Active Motif # 39538), H3K9me3 (Abcam #ab8898), LAMP1 (Iowa Hybridoma Bank #H4a3-s), and Flag (Sigma-Aldrich #F1804).
9
Chromatin Immunoprecipitation and Sequencing
10
Chromatin Immunoprecipitation (ChIP) Assay
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Chromatin immunoprecipitation (ChIP) assays were performed as previously described [16 (link)]. Differences in DNA enrichment for ChIP samples were determined by qPCR using 2.5% of the precipitated sample DNA and 0.02% of the input DNA. PCR primers used for ChIP are available upon request. Briefly, cells were cross-linked with 1% formaldehyde for 10 minutes and incubated for 5 minutes at room temperature with 0.125 M glycine to terminate the reaction. Nuclei were prepared, digested with 50 U micrococcal nuclease for 15 minutes at 37°C, and sonicated to yield chromatin fragments (200-400 bp). The precleared chromatin was incubated overnight at 4°C with H3K4me3 (07-473, Millipore, Billerica, MA), H3K27me2 (07-452, Millipore), H3K9me3 (Ab8898, Abcam, Cambridge, MA), followed by incubation with protein A agarose (Millipore), which was pre-equilibrated with sonicated salmon sperm DNA and bovine serum albumin. Immunoprecipitated material was washed extensively, and the crosslinks were reversed. DNA from the eluted chromatin was purified by phenol extraction and ethanol precipitation.
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