Chromatin immunoprecipitation (ChIP) was performed as previously described with some necessary modification (Liu et al., 2019 (link)). B. cinerea strains were grown on potato dextrose agar (PDA) slants at 25°C for 6 days. Mycelia were collected in filtered and clarified 1xPDB liquid medium with 1% (v/v) formaldehyde. Crosslinking was performed at 25°C and 90 rpm for 15 min, then 125 mM glycine was added to terminate fixation, and incubated at room temperature for 5 min after mixing. Subsequently, the treated samples were rinsed with water, frozen and ground in liquid nitrogen for chromatin isolation. Anti-H3K9me3 (Active Motif) and anti-H3K14ac (Active Motif) were used for immunoprecipitation into the chromatin. Immunoprecipitated DNA was used for ChIP followed by sequencing (ChIP-seq) DNA library preparation or ChIP-qPCR analysis.
Anti h3k9me3
Manufactured by Active Motif
Sourced in United States
Anti-H3K9me3 is a laboratory reagent designed to detect and quantify the presence of trimethylation at lysine 9 of histone H3 (H3K9me3), a post-translational modification associated with heterochromatin formation and transcriptional silencing. This antibody can be used in various applications, such as chromatin immunoprecipitation (ChIP), western blotting, and immunofluorescence, to study epigenetic regulation and chromatin dynamics.
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Lab products found in correlation
Anti h3k9me2, by Active Motif (7 mentions)
Anti h3k27me3, by Active Motif (7 mentions)
Anti h3, by Abcam (4 mentions)
Anti h3k27me3, by Merck Group (4 mentions)
Anti h3k4me3, by Abcam (3 mentions)
Anti flag, by Merck Group (3 mentions)
Anti igg, by Santa Cruz Biotechnology (3 mentions)
Chip it express kit, by Active Motif (2 mentions)
Anti h3, by Active Motif (2 mentions)
Anti akt1, by Cell Signaling Technology (2 mentions)
Secondary anti rabbit and or anti mouse antibodies, by Merck Group (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Ecl plus, by GE Healthcare (2 mentions)
Anti h3k36me3, by Abcam (2 mentions)
Las af, by Leica camera (2 mentions)
Anti mouse alexa 488, by Jackson ImmunoResearch (2 mentions)
Mouse anti gad67, by Abcam (2 mentions)
Anti mouse alexa 546, by Jackson ImmunoResearch (2 mentions)
Draq5, by Enzo Life Sciences (2 mentions)
Mouse anti neun, by Merck Group (2 mentions)
34 protocols using anti h3k9me3
1
ChIP-seq Profiling of Histone Marks
2
Chromatin Immunoprecipitation of SETDB2
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MKN74 and MKN45 cells were transfected with SETDB2 siRNA or negative control siRNA for 48 hours. ChIP assays were performed using the ChIP-IT Express Kit (Active Motif) according to the manufacturer's instructions [9 (link)]. AGS cells were transfected with the SETDB2/pcDNA3 or empty/pcDNA3 expression vector. Antibodies used were anti-H3 (Active Motif), anti-H3K9me3 (Active Motif), Normal Rabbit IgG (No. 2729, Cell Signaling Technology, Danvers, MA) and anti-SETDB2 (No. 5517, abcam).
3
Histone Modifications in Brain Regions
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Frozen tissue punches of the relevant regions were homogenized and boiled in 1% SDS. For clozapine and quinpirole treatments tissues were obtained 24h after the last injection. Extracts (50 μg) were separated on SDS-PAGE and transferred onto membranes. Antibody used were: anti-H3K9me2 (1:5000), anti-H3K9me3 (1:1000) from Active Motif; anti-H3K4me3 (1:1000), anti-H3 (1:1000) from Abcam; anti-GAPDH (1:20,000) Millipore; anti-Akt1 (1:1000) Cell Signaling; anti-NR4A2 (1:1000) Antibody Verify. Secondary anti-rabbit and/or anti-mouse antibodies (1:5000) were from Millipore. Western blots were revealed using ECL Plus (GE Healthcare or Westdura, Pierce). Quantifications were performed using the NIH ImageJ (version 1.42q) software.
4
Protein Analysis by Western Blot
5
Chromatin Profiling in Metaphase Cells
6
Quantification of H3K9me3 in Brain Sections
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Immunostainings were performed as described in SM on 10 μm coronal brain sections using rabbit anti-H3K9me3 (1:500, Active Motif), mouse anti-GAD67 (1:500, Abcam), and mouse anti-NeuN (1:500, Millipore) antibodies followed by anti-rabbit CY3 (1:500, Jackson laboratories) or anti-mouse Alexa-488 (1:800, Jackson Laboratories) or anti-mouse Alexa-546 (1:500, Jackson Laboratories) antibodies. Nuclei were counterstained with Draq5 (1:500, Enzo Life Science). Images were taken at the confocal (DMRE; SP5 Leica) and quantified using LAS AF (Leica) in 3 regions of interest (ROI) of 145x145 μm/image (in Fig. 2f n=16-18 images/mouse 3mice/genotype; in Fig. 3f n=14 images/mouse 3mice/genotype). Data represent ratios of pixels fluorescence/Draq5 per ROIs.
7
Visualizing Histone Modifications in Embryos
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To visualize H3K9me3 and H4K20me1, we stained fixed embryos with primary antibodies (rabbit anti-H3K9me3 and mouse anti-H4K20me1, Active Motif Inc.) diluted at 1:500 in 1× PBT overnight at 4°C on a platform rocker. Embryos were then washed three times at 10 min each with 1× PBT and then stained with fluorescently conjugated secondary antibodies at room temperature for 1 hour on a platform rocker in the dark. Secondary antibodies used in this study were anti-rabbit Cy3 and anti-mouse Cy5 (both at 1:300; Invitrogen, Thermo Fisher Scientific Inc., USA). Embryos were then washed as stated above and then mounted on a slide with VECTASHIELD mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc., USA).
8
Chromatin Immunoprecipitation of H3K9me3
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Nuclear lysate was sonicated to make small DNA fragments ranging from 100–500 base pairs and then incubated with anti-H3K9me3 (MABI 0319, Active motif, Carlsbad, CA) overnight at 4°C. Isotype-matched control Ab was used for the negative control. Immune complexes containing DNA fragments were precipitated using EZ-ChIP kit (Milipore, Darmstadt, Germany). Relative enrichment of the target regions in the precipitated DNA fragments was analyzed by qPCR. The sequences of primers are as follows.
9
Antibodies for Chromatin Immunoprecipitation
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The primers and synthetic DNAs used in this study are listed in Supplementary file 1 . The following antibodies were obtained from manufacturers: anti-H3K27me3 (Millipore, 07–449, RRID:AB_310624 ); anti-HA HA11 (Covance, clone 6B12, RRID:AB_291231 ); anti-H3K9me3 (Active Motif, 39162); anti-GST (BD Biosciences, 554805, RRID:AB_395536 ); anti-His (Proteintech, 66005–1-Ig, RRID:AB_11232599 ); anti-long dsRNA J2 (Jena Bioscience RNT-SCI-10010200, RRID:AB_2651015 ); anti-α-tubulin 12G10 (Developmental Studies Hybridoma Bank of the University of Iowa). The anti-Rpb3 antibody was described previously (Kataoka and Mochizuki, 2017 (link)). The anti-Smt3 antibody was a gift from Dr. James Forney (Purdue University) and described previously (Nasir et al., 2015 (link)).
10
Cell Culture and Protein Analysis
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NB4, HL60, K562, Jurkat, and U937 cell lines were maintained in RPMI 1640 medium supplemented with 10% of fetal bovine serum (FBS, Gibco, Paisley, UK), 1% of L-glutamine (Lonza, Verviers, Belgium) and 1% of penicillin/streptomycin (Euroclone, Pero, MI, Italy) in a humidified atmosphere at 37 °C and 5% of CO2. Cell lines were originally obtained from ATCC repository and routinely tested by PCR method and MycoAlert (Lonza, Verviers, Belgium #LT07-318) for mycoplasma contamination by the European Institute of Oncology (Milan, Italy).
Maltonis was synthesized as already described [20 (link)] and used for the treatments of cells (stock solution of 10 mM diluted in distilled water) at the reported concentrations for 24 h [19 (link)]. Western blot analyses were performed as previously reported [23 (link)] using the following antibodies: anti-H3K9me3 (#39766, Active Motif, Carlsbad, CA, USA, 1:1000 dilution), anti-α-tubulin (#T9026, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany, 1:500 dilution), anti-Histone H3 (#05-499, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany, 1:500 dilution), anti-c-MYC (Y69, Abcam, Cambridge, MA, USA, 1:1000 dilution), and images acquired using Vü-C Imaging system (PopBio, Cambridge, UK).
Maltonis was synthesized as already described [20 (link)] and used for the treatments of cells (stock solution of 10 mM diluted in distilled water) at the reported concentrations for 24 h [19 (link)]. Western blot analyses were performed as previously reported [23 (link)] using the following antibodies: anti-H3K9me3 (#39766, Active Motif, Carlsbad, CA, USA, 1:1000 dilution), anti-α-tubulin (#T9026, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany, 1:500 dilution), anti-Histone H3 (#05-499, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany, 1:500 dilution), anti-c-MYC (Y69, Abcam, Cambridge, MA, USA, 1:1000 dilution), and images acquired using Vü-C Imaging system (PopBio, Cambridge, UK).
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