Histone h3 antibody

Manufactured by Cell Signaling Technology

Sourced in United States

The Histone H3 antibody is a primary antibody that targets the histone H3 protein, which is a core component of nucleosomes in eukaryotic cells. This antibody can be used to detect and quantify the presence of histone H3 in various biological samples, such as cell lysates or tissue extracts, using techniques like Western blotting or immunohistochemistry.

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Lab products found in correlation

Bovine serum albumin (bsa), by Merck Group (3 mentions) Chip grade protein g magnetic beads, by Cell Signaling Technology (2 mentions) Hotstartaq master mix, by Qiagen (2 mentions) Acetyl histone h3 lys9, by Cell Signaling Technology (2 mentions) Catalase, by Santa Cruz Biotechnology (1 mentions) Quercetin dihydrate, by Merck Group (1 mentions) Rhodamine 123, by Merck Group (1 mentions) Agarose, by Merck Group (1 mentions) Phospho jnk, by Santa Cruz Biotechnology (1 mentions) Anti β actin antibody, by Merck Group (1 mentions) Phospho p38, by Santa Cruz Biotechnology (1 mentions) Phospho mek, by Santa Cruz Biotechnology (1 mentions) Mek1 2, by Santa Cruz Biotechnology (1 mentions) Phospho erk, by Santa Cruz Biotechnology (1 mentions) Cu zn sod, by Santa Cruz Biotechnology (1 mentions) Phospho akt, by Santa Cruz Biotechnology (1 mentions) Erk1 2, by Santa Cruz Biotechnology (1 mentions) Fluo 3 am, by Merck Group (1 mentions) Ribonuclease a, by Merck Group (1 mentions) Propidium iodide, by Merck Group (1 mentions)

Close spelling variants of this product

Anti-p-Histone H3 (Ser 10) antibody (3 citations) Anti‐Histone H3 monoclonal antibody (2 citations) Anti‐histone H3 antibody (positive control, (2 citations) Acetyl-histone H3 antibody sampler kit (4 citations) Alexa Fluor 488 conjugated phospho-Histone H3 (Ser10) antibody (DC28, (2 citations) Anti-histone H3 antibody (31 citations) Acetyl-histone H3 (Lys9/Lys14) antibody (3 citations) Alexa Fluor-488 conjugated phosphorylated histone H3 antibody (2 citations) Phospho-histone H3 (Ser10) antibody (11 citations) Acetyl-histone H3 antibody (5 citations)

23 protocols using histone h3 antibody

Quercetin Modulation of Cell Signaling

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Dulbecco’s modified Eagle’s media (DMEM), Dulbecco’s phosphate buffer saline (DPBS), fetal bovine serum (FBS), penicillin–streptomycin, quercetin dihydrate (Qu), 3-(4,5-dimetylthiazol-yl)-diphenyl tetrazolium bromide (MTT), propidium iodide, rhodamine 123, 2,7-dichlorodihydrofluoresceindiacetate (H₂DCF-DA), Fluo-3 AM, proteinase K, ribonuclease A, 1,2-bis-(O-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), β-nicotinamide adenine dinucleotide reduced disodium salt (β-NADH), ascorbic acid, agarose, dimethyl sulfoxide (DMSO) and anti-β-actin antibody were purchased from Sigma–Aldrich Chemicals (St. Louis, MO). Antibodies against Bcl-2, Bax, Bim, AIF, NF-κB, caspase-3/caspase-8, phospho-MEK, MEK1/2, phospho-ERK, ERK 1/2, phospho-p38, p-38, phospho-JNK, JNK, Nrf-2, catalase, Cu/Zn SOD, PI3K, phospho-Akt, Akt and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc,). FITC Annexin V was purchased from BD Pharmingen. Histone H3 antibody was purchased from Cell Signaling Technology.
Stock solution of quercetin (Qu) was prepared in dimethyl sulfoxide (DMSO) and diluted to the desired final concentration with culture medium just before use. The final DMSO concentration did not exceed 0.1% (v/v).

Rafiq R.A., Quadri A., Nazir L.A., Peerzada K., Ganai B.A, & Tasduq S.A. (2015). A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells. PLoS ONE, 10(7), e0131253.

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Breast Cancer Cell Lines Characterization

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ER- human breast non-tumorigenic epithelial cell lines (MCF-10A, MCF-10F and MCF-10-2A), ER+ human breast cancer cell lines (MCF-7 and T-47D) and ER- human breast cancer cell lines MDA-MB231 and SKBR-3) were from ATCC (Manassas, Virginia, USA). Tamoxifen was from Sigma-Aldrich. Cell culture medium (DMEM/F12), OPTI-MEM, Lipofectamine 2000 and TRIzol reagent were from Life Technologies (San Diego, CA, USA). Antibodies against β-actin and TFIIIC₆₃ and c-Jun siRNA (Catalog No. SC-29224) were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Mismatch RNA was described previously [24 (link)]. Histone H3 antibody were from Cell Signaling (Danvers, MA, USA). Brf1 antibody was from Bethyl laboratories Inc (Montgomery, TX, USA). The sequences of primers were described in (Supplements) [8 (link), 30 (link)].

Zhong Q., Shi G., Zhang Q., Lu L., Levy D, & Zhong S. (2014). Tamoxifen represses alcohol-induced transcription of RNA polymerase III-dependent genes in breast cancer cells. Oncotarget, 5(23), 12410-12417.

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Evaluating WEE1 and Phospho-cdc2 Expression

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The WEE1 and Phospho-cdc2 (Tyr15) expressions were examined by Western blotting. A nuclear protein and plasma protein extraction kit (Generay, Shanghai, China) was used to extract nuclear protein, which was separated using 11% polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto the top of the polyvinylidene fluoride (PVDF) membrane; PBS (PH 7.2) containing 6% dried skim milk was used to block at room temperate for 1–2 h; afterwards, the blocked PVDF membrane was placed in a freezer at 4°C overnight. Wee1 antibody and Tyr15 antibody (all at 1:500 dilution; Cell Signaling Technology) and Histone H3 antibody (at 1:1000 dilution; Cell Signaling Technology) were add as the primary antibody. After staying at room temperate for 1 h, it was washed with PBS, and human antibody against rabbit antibiotin-horseradish peroxidase (HRP) -IgG at 1:2500 dilution was added. At the same time, 40 μL of HRP antibodies at 1: 1000 dilution was added for reaction at room temperate for 1 h. Then, chemiluminescence reagent was added and immunoautoradiography was used to assess protein expressions.

Lou W., Zhang X., Hu X.Y, & Hu A.R. (2016). MicroRNA-219-5p Inhibits Morphine-Induced Apoptosis by Targeting Key Cell Cycle Regulator WEE1. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 22, 1872-1879.

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ChIP Assay Protocol for ZBTB48 Binding

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ChIP assays were performed using the ChIP Assay Kit (Beyotime) according to the manufacturer’s instructions. Briefly, an anti-ZBTB48 antibody, Histone H3 antibody (Cell signaling technology) as a positive control and IgG antibody (Abcam) as a negative control were used to immunoprecipitate the chromatin complexes. Then DNA was extracted and purified from the complexes and analyzed by qPCR. The primers for ChIP qPCR are listed in Table S5.

Wang C., Zhang M., Liu Y., Cui D., Gao L, & Jiang Y. (2023). CircRNF10 triggers a positive feedback loop to facilitate progression of glioblastoma via redeploying the ferroptosis defense in GSCs. Journal of Experimental & Clinical Cancer Research : CR, 42, 242.

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Preparation of Nuclear Extracts from Liver Tissue

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Nuclear extracts were prepared using a slight modification of the method described by Dignam, Lebovitz & Roeder (1983) (link). Briefly, samples of frozen liver tissue (∼1 g) were homogenized in 1 mL of homogenization buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 10 mM EDTA) with the addition of 10 µL of 100 mM dithiothreitol (DTT) and 10 µL protease inhibitor cocktail. Samples were centrifuged at 10,000 rpm for 10 min at 4 °C and the supernatant (cytoplasmic extract) was removed. The pellet was resuspended in 147 µL of extraction buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 10% v:v glycerol) with 1.5 µL of 100 mM DTT and 1.5 µL of protease inhibitor cocktail added. The tubes were capped, put in ice on a rocking platform for 1 h, and then centrifuged at 10,000× g for 10 min at 4 °C. The supernatant (nuclear extract) was collected. The integrity of the nuclei was confirmed by immunoblotting of cytoplasmic and nuclear fractions and probing with a histone H3 antibody (Cell Signaling; Cat# 9715).

Biggar K.K, & Storey K.B. (2018). The evaluation of anoxia responsive E2F DNA binding activity in the red eared slider turtle, Trachemys scripta elegans. PeerJ, 6, e4755.

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ChIP Assay Protocol Using SimpleChIP Kit

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ChIP assay was conducted by using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, USA) in accordance with the protocol formulated by the manufacturer. In brief, the cells were cross-linked with formaldehyde, lysed with sodium dodecyl sulfate buffer, and then sonicated. Following sonication, the fragmented chromatin was added into ChIP dilution buffer and incubated overnight with primary antibody. IgG antibody (Cell Signaling Technology) was taken as a negative control and Histone H3 antibody (Cell Signaling Technology) was taken as a positive control. Immunoprecipitation product was gathered following the incubation with ChIP-Grade Protein G Magnetic Beads (Cell Signaling Technology). After the binding chromatin was eluted and digested with Proteinase K (Cell Signaling Technology), the purified DNA was applied to conduct qRT-PCR assay. Supplemental Table S2 lists the antibodies used for ChIP and Supplementary Table S4 shows the sequences of forward and reverse primers used for ChIP.

Li H., Zheng N., Guo A., Tang W., Li M., Cao Y., Ma X., Cao H., Ma Y., Wang H, & Zhao S. (2024). FSTL3 promotes tumor immune evasion and attenuates response to anti-PD1 therapy by stabilizing c-Myc in colorectal cancer. Cell Death & Disease, 15(2), 107.

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Chromatin Immunoprecipitation (ChIP) of MEIS1

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Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell Signaling #9005). Briefly, 12 × 10⁶ cells were collected and fixed with 1.5% of fresh formaldehyde, incubated for 10 min at 20 °C, and stopped with 125 mM glycine for 5 min at 4 °C. Next, the SimpleChIP^® Plus Enzymatic Chromatin IP protocol was followed. The Adaptive Focused Acoustics™ (AFA) Technology from Covaris (Waltham, MA, USA) was used to sonicate chromatin for 4 min with 5% of sonication (75 Watts) two times. Chromatin was precleared with protein G agarose beads (Cell Signaling Technology, Waltham, MA, USA) for 2 h at 4 °C and immunoprecipitated with 8.5 μL of MEIS1-specific antibodies (Atlas Antibodies HPA056000), a negative control IgG antibody, or a positive control Histone H3 antibody (Cell Signaling Technology) overnight at 4 °C.
PCRs were performed in triplicate using the HotStarTaq Master Mix (QIAGEN), and the primer sequences used are listed in Table S5.
All immunoprecipitations were carried out in triplicate, using different chromatin preparations.

Le Nabec A., Blotas C., Briset A., Collobert M., Férec C, & Moisan S. (2022). 3D Chromatin Organization Involving MEIS1 Factor in the cis-Regulatory Landscape of GJB2. International Journal of Molecular Sciences, 23(13), 6964.

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ChIP Assay for Hepatocyte Chromatin

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Chromatin was prepared from hepatocytes for ChIP assays as previously described. Cells were fixed with 4% paraformaldehyde for 15 min, then glycine was added to a final concentration of 0.125 M and incubated for 10 min before harvesting. Chromatin was sonicated using a Bioruptor Pico instrument (Diagenode, Denville, NJ, USA). Chromatin preparations were subjected to ChIP using a ChIP-IT High Sensitivity Kit and Protein G Agarose Prepacked Columns (Active Motif, Carlsbad, CA, USA) using a PPARα (Abcam Ab24509) antibody. Normal rabbit IgG (Cell Signaling Technologies #2729S) and Histone H3 antibody (Cell Signaling Technologies #4620) were used as negative and positive controls, respectively. DNA was purified and concentrated using MinElute Reaction Cleanup columns (Qiagen). qRT-PCR and conventional PCR were performed using 2 μl of ChIP DNA samples from the 50 μl of purified samples using gene-specific primers (Table S1). Cycle threshold (Ct) values of ChIP and input samples were calculated and presented as fold change values.

Kim D., Kim B., Brocker C.N., Karri K., Waxman D.J, & Gonzalez F.J. (2022). Long non-coding RNA G23Rik attenuates fasting-induced lipid accumulation in mouse liver. Molecular and cellular endocrinology, 557, 111722.

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Evaluating Tazemetostat Effects on EZH2

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At the end of the tazemetostat treatment, we collected fresh tumors and peripheral blood mononuclear cells (PBMC) to assess the efficacy of the drug on EZH2 activity. PBMCs were exposed to RBC lysis buffer (NH₄Cl: 155 mM; NaHCO₃: 10 mM; EDTA: 0.1 mM) for 5 min on ice and washed with MACS buffer twice to remove red blood cells. Subsequently, PBMC were labeled with Aqua dead (L34966A; Invitrogen) for 30 min at 4 °C in PBS and subsequently fixed with fixation/permeabilization buffer (Ref: 00-5223-56, Invitrogen) during 30 min at 4 °C. Afterward, cells were intracellularly labeled with histone H3 antibody (ref: 12230S; Cell Signaling) and the specific H3K27me3 antibody (reference: 5499S) for 30 min at 4 °C in permeabilization buffer (Ref: 00-8333-56, Invitrogen). Finally, cells were washed and resuspended in MACs buffer for FACS analysis in BD LSR Fortessa. Gates were defined on single and live cells and the signal of H3K27me3 staining was calculated as a ratio of total H3 staining. In parallel, freshly resected tumors were fixed in formol/ethanol, and then embedded in paraffin; H3K27me3 staining was assessed using the tri-methyl-histone H3 (Lys27) (C36B11) with the previously described procedure. Brain delivery was not evaluated considering the extradural bone origin of chordomas.

Passeri T., Dahmani A., Masliah-Planchon J., Naguez A., Michou M., El Botty R., Vacher S., Bouarich R., Nicolas A., Polivka M., Franck C., Schnitzler A., Némati F., Roman-Roman S., Bourdeaut F., Adle-Biassette H., Mammar H., Froelich S., Bièche I, & Decaudin D. (2022). Dramatic In Vivo Efficacy of the EZH2-Inhibitor Tazemetostat in PBRM1-Mutated Human Chordoma Xenograft. Cancers, 14(6), 1486.

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Cytoplasmic and Nuclear Protein Extraction

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Nuclear and cytoplasmic extracts were made according to previously published protocols (Smith et al., 2004 (link); Rozenfeld and Devi, 2007 (link)). Briefly, PANC-1 cells were treated with 10 nM dermorphin or 10 nM (1R,1′S,3′R/1R,1′R,3′S)-L-054,264 (Tocris, Bristol, UK) individually and in combination for the indicated times. After the treatment, cells were washed in ice-cold PBS three times and scraped into lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 1 mM ethylene glycol tetraacetic acid, 1 mM sodium orthovanadate, and a protease phosphatase inhibitor tablet [ThermoFisher Pierce]) and incubated on ice for 10 min. The lysate was then homogenized and centrifuged at 375 × g for 5 min. The pellet consisting of the nuclear fraction was washed five times with lysis buffer containing 0.1% NP-40 to remove any nonnuclear contamination and resuspended in lysis buffer containing NP-40. The soluble fraction was centrifuged twice at 375 × g to remove nuclear contamination and used as the cytosolic fraction. Fractions were then used for immunoblotting for anti–phospho-ERK1/2, anti–phospho-p90RSK (Thr-573; rabbit polyclonal; Cell Signaling, Danvers, MA), and anti–β-actin as a loading control for the cytoplasmic fraction, and Histone H3 antibody (rabbit monoclonal; Cell Signaling) was used as a loading control for the nuclear fraction.

Jorand R., Biswas S., Wakefield D.L., Tobin S.J., Golfetto O., Hilton K., Ko M., Ramos J.W., Small A.R., Chu P., Singh G, & Jovanovic-Talisman T. (2016). Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer. Molecular Biology of the Cell, 27(22), 3659-3672.

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