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Histone h3

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Histone H3 is a core histone protein found in eukaryotic cells. It is involved in the structural organization of chromatin by forming nucleosomes, the fundamental units of chromatin.

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Lab products found in correlation

β actin, by Abcam (32 mentions) β actin, by Merck Group (28 mentions) Gapdh, by Abcam (20 mentions) Flag tag (flag), by Merck Group (20 mentions) β actin, by Cell Signaling Technology (15 mentions) Gapdh, by Cell Signaling Technology (12 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions) Pvdf membrane, by Merck Group (11 mentions) Gapdh, by Santa Cruz Biotechnology (10 mentions) Gapdh, by Merck Group (9 mentions) Vimentin, by Abcam (8 mentions) Histone h4, by Abcam (8 mentions) β actin, by Santa Cruz Biotechnology (8 mentions) Flag m2, by Merck Group (7 mentions) H3k4me3, by Abcam (7 mentions) α tubulin, by Santa Cruz Biotechnology (7 mentions) Nf κb p65, by Cell Signaling Technology (7 mentions) Protease inhibitor cocktail, by Roche (7 mentions) E cadherin, by Abcam (6 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (6 mentions)

Close spelling variants of this product

Rabbit anti-Histone H3 (K27Ac) Anti-phospho-histone H3 (Ser10; Anti-histone H3-acetyl K18 Rabbit anti-histone H3 (acetyl K27) Anti-Histone H3 trimethylated on Lysine 4 (H3K4me3) Citrullinated histone H3 Anti–trimethyl-Histone H3 Lys4 Phosphorylated histone H3 (phospho-S10, Histone H3 trimethylK36 Histone H3 (acetyl K9)

263 protocols using histone h3

1

Subcellular Fractionation and Molecular Analyses

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Western blot and quantitative RT-qPCR (RT-qPCR) of marker proteins/transcripts for HEK293-subcellular compartments were used to verify the subcellular fractionation results. Western blots were performed with antibodies for three proteins – GAPDH (Abcam), SNRP70 (Abcam) and histone H3 (Abcam). Samples of subcellular fractions were boiled at 95°C for 10 minutes, then spun at 14000g for 3 minutes at room temperature to minimize the influence of sticky DNA (especially in the chromatin samples) on Western blots. For every Western blot, 1% of the sample volume was used. For RT-qPCR, the same percentage (1%) of RNA samples was used. Then the ratio of each marker gene (GAPDH, U1, ACTIN [intron]) was calculated in the respective chromatin, nucleoplasmic and cytoplasmic fractions.
For mES cells, similar western-blot experiments were carried out using antibodies against actin (Abcam), histone H3 (Abcam) and SNRP70 (Abcam). We confirmed NAI-N3 treatment didn’t affect fractionation. Protein was quantified using the Pierce BCA protein assay kit (Thermo Fischer Scientific). We loaded the protein from each compartment in proportion to the amount obtained (roughly 1:1:2 for chromatin:nucleoplasm:cytoplasm).
Sun L., Fazal F.M., Li P., Broughton J.P., Lee B., Tang L., Huang W., Kool E.T., Chang H.Y, & Zhang Q.C. (2019). RNA structure maps across mammalian cellular compartments. Nature structural & molecular biology, 26(4), 322-330.
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2

Subcellular Fractionation and Molecular Analyses

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Western blot and quantitative RT-qPCR (RT-qPCR) of marker proteins/transcripts for HEK293-subcellular compartments were used to verify the subcellular fractionation results. Western blots were performed with antibodies for three proteins – GAPDH (Abcam), SNRP70 (Abcam) and histone H3 (Abcam). Samples of subcellular fractions were boiled at 95°C for 10 minutes, then spun at 14000g for 3 minutes at room temperature to minimize the influence of sticky DNA (especially in the chromatin samples) on Western blots. For every Western blot, 1% of the sample volume was used. For RT-qPCR, the same percentage (1%) of RNA samples was used. Then the ratio of each marker gene (GAPDH, U1, ACTIN [intron]) was calculated in the respective chromatin, nucleoplasmic and cytoplasmic fractions.
For mES cells, similar western-blot experiments were carried out using antibodies against actin (Abcam), histone H3 (Abcam) and SNRP70 (Abcam). We confirmed NAI-N3 treatment didn’t affect fractionation. Protein was quantified using the Pierce BCA protein assay kit (Thermo Fischer Scientific). We loaded the protein from each compartment in proportion to the amount obtained (roughly 1:1:2 for chromatin:nucleoplasm:cytoplasm).
Sun L., Fazal F.M., Li P., Broughton J.P., Lee B., Tang L., Huang W., Kool E.T., Chang H.Y, & Zhang Q.C. (2019). RNA structure maps across mammalian cellular compartments. Nature structural & molecular biology, 26(4), 322-330.
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3

Quantification of Protein Expression Levels

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Protein extraction was performed according to the instructions provided by the manufacturer of the Protein Extraction Kit (Beyotime Biotech Inc., Nantong, China). Protein extracts were separated by 10 or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked with 5% nonfat milk for 2 h, and then incubated overnight at 4 °C with Nrf2 (1:1000, Abcam, Cambridge, MA, USA), NF-κB, IL-6, IL-1β (1:400, Cell Signaling Technology, Danvers, MA, USA), histone H3 (1:1000, Abcam, Cambridge, MA, USA), β-actin (1:5000, Bioworld Technology, St. Louis Park, MN, USA), Bax (1:400, Abcam, Cambridge, MA, USA), Bcl-2 (1:200, Santa Cruz, Santa Cruz, CA, USA), histone H3 (1:1000, Abcam, Cambridge, MA, USA), and cleaved caspase-3 (1:1000, Cell Signaling Technology, Danvers, MA, USA). Subsequently, the membranes were incubated for 2 h with corresponding secondary antibodies conjugated with horseradish peroxidase (1:1000, Bioworld Technology, MN, USA) at room temperature for 2 h. The protein bands were exposed to Tanon-5200 Chemiluminescent Imaging System and strip gray levels were quantified by software (version 4.62; Bio-Rad Laboratories Inc., Berkeley, CA, USA).
Fu C., Xin H., Qian Z., Li X., Gao J., Fan Y., Tang Y., Shi Y., Li D, & Wu H. (2023). Sinomenine Protects against Early Brain Injury by Inhibiting Microglial Inflammatory Response via Nrf2-Dependent Pathway after Subarachnoid Hemorrhage. Brain Sciences, 13(5), 716.
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4

Molecular Mechanisms of MGCD-Induced Apoptosis

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MGCD was kindly provided by ROCHE R & D CENTER (CHINA) LTD. Nutlin-3 was purchased from Sigma. For western blot detection, we used Ac-Histone H3 (Upstate), Histone H3, Cleaved caspase-3, Bcl-2, Cyclin B1, Bax (Epitomics), p-cdc2 (Tyr15), cdc2, p53, p21 (Cell Signaling Technology), cleaved PARP (Santa Cruz), and GAPDH, tubulin (Proteintech Group) antibodies.
Yan M., Qian Y.M., Yue C.F., Wang Z.F., Wang B.C., Zhang W., Zheng F.M, & Liu Q. (2016). Inhibition of histone deacetylases induces formation of multipolar spindles and subsequent p53-dependent apoptosis in nasopharyngeal carcinoma cells. Oncotarget, 7(28), 44171-44184.
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5

Klotho Protein Expression and Function

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Recombinant soluble human klotho protein (sKL) was obtained from PeproTech (Rocky Hill, NJ, USA). Antibodies used were β-catenin, cyclin Dl, c-myc, histone H3, β-actin (Epitomics, USA), and klotho (Abcam, USA). Small interfering RNA oligos for knocking down klotho (siRNA-kl) and non-target control siRNA (siRNAc) were purchased from Santa Cruz Biotechnology. An expression plasmid encoding klotho (pCMV6-KL) and the control vector pCMV6-AC-GFP (pCMV6) were purchased from OriGene Technologies (Rockville, MD, USA). All plasmid constructs were confirmed by DNA sequencing.
Tang X., Wang Y., Fan Z., Ji G., Wang M., Lin J., Huang S, & Meltzer S.J. (2015). Klotho: a tumor suppressor and modulator of the Wnt/β-catenin pathway in human hepatocellular carcinoma. Laboratory investigation; a journal of technical methods and pathology, 96(2), 197-205.
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6

Comprehensive Antibody Panel for Western Blotting

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Primary antibodies: Actin (Millipore, MAB1501, 1:10 000), DAXX (Sigma-Aldrich, D7810-2ML, 1:1000), DAXX (Cell Signaling Technology, #4533, 1:1000), ATRX (Abcam, ab97508, 1:1000), GAPDH (Cell Signaling Technology, #5174, 1:7000), RNase H1 (Abcam, ab229078, 1:1000), BRCA1 (Bethyl, A300-000A, 1:1000), GFP (Roche, 11814, 1:1000), Vinculin (Santa Cruz, sc-73614, 1:2000), HSP60 (Sigma-Aldrich, H3524, 1:2000), KAP1 (Abcam, ab10483,1:1000), SETDB1 (Proteintech,11231–1-AP,1:1500), Histone H3.3 (Abcam, ab176840, 1:1000), Histone H4 (Cell Signaling Technology, #13919, 1:1000). Secondary antibodies: HRP rabbit (Jackson Laboratory, Cat. No. 111-035-003, 1:50 000), HRP mouse (Amersham/Sigma, Cat. No. GENA931-1ML, 1:10 000).
Pinto L.M., Pailas A., Bondarchenko M., Sharma A.B., Neumann K., Rizzo A.J., Jeanty C., Nicot N., Racca C., Graham M.K., Naughton C., Liu Y., Chen C.L., Meakin P.J., Gilbert N., Britton S., Meeker A.K., Heaphy C.M., Larminat F, & Van Dyck E. (2023). DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks. Nucleic Acids Research, 52(3), 1136-1155.
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7

Immunoblotting and Immunofluorescence Analyses

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The following antibodies and probes were used where indicated. ATRX (Cell Signaling, D1N2E), ATRX (Santa Cruz H-300), DAXX (Cell Signaling 25C12), FLAG-M2 (Sigma F1804), GAPDH (Santa Cruz, 0411), Histone H3.3 (Abcam, EPR17899), Histone H4 (Active Motif, 39269), PML (Santa Cruz H-238), PML (Santa Cruz PG-M3), α-Tubulin (Cell Signaling, 11H10), Purified Rabbit IgG (Bethyl Laboratories P120-101), Alexa Fluor 488 conjugated Donkey Anti-Rabbit IgG (Jackson ImmunoResearch), Cy3 conjugated Donkey anti-Rabbit (Jackson ImmunoResearch), Peroxidase conjugated Goat Anti-Mouse IgG (Jackson ImmunoResearch), Peroxidase conjugated Goat Anti-Rabbit IgG (Jackson ImmunoResearch). PNA Telomere probe (TelC-Cy3, PNA Bio Inc.) Flag-DAXX/pRK5 was a gift from Xiaolu Yang (Addgene plasmid #27974).
Mason-Osann E., Dai A., Floro J., Lock Y.J., Reiss M., Gali H., Matschulat A., Labadorf A, & Flynn R.L. (2018). Identification of a novel gene fusion in ALT positive osteosarcoma. Oncotarget, 9(67), 32868-32880.
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8

DAXX Chromatin Immunoprecipitation Protocol

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We prepared sonicated DNA samples from DAXX-WT and DAXX-KO cells using a ChIP-IT Express (No. 39163, Active Motif, Carlsbad, CA, USA), and then incubated with 1-2 μg of three antibodies; DAXX (1 μg, sc7152; rabbit polyclonal, Santa Cruz Biotechnology), Histone H3.3 (2 μg, ab176840; rabbit monoclonal, Abcam) and Histone H3K9me3 (1 μg, 39765, Active Motif). Normal rabbit IgG (Cell Signaling Technology) was used as a negative control for each assay. Input DNA samples were used as internal controls. The primer sequences and their conditions are shown in Supplementary Table 1.
Ueda H., Akiyama Y., Shimada S., Mogushi K., Serizawa M., Matsumura S., Mitsunori Y., Aihara A., Ban D., Ochiai T., Kudo A., Tanabe M, & Tanaka S. (2018). Tumor suppressor functions of DAXX through histone H3.3/H3K9me3 pathway in pancreatic NETs. Endocrine-related cancer, 25(6).
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9

Western Blot Analysis of Prostate Proteins

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Protein was extracted from whole-cell lysates using Kinexus Lysis Buffer with Lysis Buffer Cocktail (Kinexus Bioinformatics Corporation, Vancouver, British Columbia, Canada) and protein concentration was determined using BCA assay (Thermo Scientific, Waltham, MA, USA). Subsequently, 30 μg of total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated with antibodies against PRMT6, H3R2me2a (1/500 Novus Biologicals, Littleton, CO), H3K4me3, PSA (1/1500 and 1/6000 Abcam, Cambridge, UK), AR, mTOR (1/1000, Cell Signaling Technology, Inc., Danvers, MA), p21, p27 (1/500, BD Biosciences, Franklin Lakes, NJ), AKT (1/500, Santa Cruz Biotechnologies Inc) and pAKT (1/500, Millipore Billerica, MA), as well as histone H3 (dilution: 1:500, Abcam) and beta-actin (dilution: 1:8000, Sigma-Aldrich) as input controls, as appropriate. Blots were developed using Immun-Star™ WesternC™ Kit (BioRad, Hercules, CA). All experiments were performed in triplicate.
Almeida-Rios D., Graça I., Vieira F.Q., Ramalho-Carvalho J., Pereira-Silva E., Martins A.T., Oliveira J., Gonçalves C.S., Costa B.M., Henrique R, & Jerónimo C. (2016). Histone methyltransferase PRMT6 plays an oncogenic role of in prostate cancer. Oncotarget, 7(33), 53018-53028.
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10

Western Blot Analysis of Cellular Proteins

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Cell lysates were run on a 4-12% gradient Bis Tris gel (Invitrogen) and transferred to a PVDF membrane using standard methods. The membrane was probed with Tubulin (Sigma #T6047; 1:1000), β-actin (Sigma #A3854; 1:14000), TIM23 (BD Biosciences #611222; 1:1000), TBK1 (Abcam #ab40676; 1:1000), phospho-TBK1 S172 (Cell Signaling #5483; 1:800), cGAS (Cell Signaling #31659; 1:800), TFEB (Abcam #ab2636; 1:800), Histone H3 (Abcam #ab1791; 1:1000). Nuclear fractions were isolated using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher #7883). Membranes were then washed and exposed (60 min) to 1:2000 HRP-conjugated secondary antibody (GE Healthcare) in 5% milk. After further washes, signal was detected with HyGLO quick spray (Thomas Scientific), followed by film exposure.
Rai P., Janardhan K.S., Meacham J., Madenspacher J.H., Lin W.C., Karmaus P.W., Martinez J., Li Q.Z., Yan M., Zeng J., Grinstaff M.W., Shirihai O.S., Taylor G.A, & Fessler M.B. (2021). IRGM1 links mitochondrial quality control to autoimmunity. Nature immunology, 22(3), 312-321.
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