H3k79me2

Manufactured by Cell Signaling Technology

Sourced in United States

H3K79me2 is an antibody that specifically recognizes the dimethylation of lysine 79 on histone H3. Histone modifications, including methylation, play a crucial role in regulating gene expression and chromatin structure.

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9 protocols using h3k79me2

Antibodies for Protein Analysis Techniques

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The following primary antibodies were used in western blotting assays: NPM1 mutant (NB110-61646, Novus, Littleton, USA), NPM1 WT (Thermo scientific, Rockford, USA), NPM1 (Sigma-Aldrich, St. Louis, USA), PBX3 (Abcam, Cambridge, USA), HOXA9 (Abcam, Cambridge, USA), Cleaved caspase 3, Cleaved PARP (Cell Signaling, Danvers, USA), Flag, H3K4me2, H3K9me2, H3K27me2, H3K36me2, H3K79me1, H3K79me2, H3K79me3, H3, DOT1L (Cell Signaling, Danvers, USA), and β-Actin (Sigma, St. Louis, MO, USA).
All antibodies and the following kits applied in flow cytometry analyses were purchased from BD Biosciences (BD Pharmingen™, New Jersey, USA): CD45.1, CD45.2, Mac-1, Gr-1, Sca-1, c-Kit, Mouse Lineage Panel, AnnexinV Apoptosis Detection kit, and BrdU Flow kit.
Immunofluorescence staining was performed with Flag (Cell Signaling, Danvers, USA), H3K79me2 (Cell Signaling, Danvers, USA and PMT-bio, Hangzhou, China), PBX3 (Santa Cruz, California, USA), HOXA9 (Abcam, Cambridge, USA) and NPM1 antibodies as following: NPM1 mutant (Novus, Littleton, USA), NPM1 WT (Thermo scientific, Rockford, USA), NPM1 (Cell Signaling, Danvers, USA).

Zhang W., Zhao C., Zhao J., Zhu Y., Weng X., Chen Q., Sun H., Mi J.Q., Li J., Zhu J., Chen Z., Pandolfi P.P., Chen S., Yan X, & Xu J. (2018). Inactivation of PBX3 and HOXA9 by down-regulating H3K79 methylation represses NPM1-mutated leukemic cell survival. Theranostics, 8(16), 4359-4371.

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Compound Dose-Dependent Protein Analysis

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3 × 10⁶ cells/well were treated with increasing concentrations of a compound for 1 day, and whole proteins were extracted. Equal amounts of proteins were separated on SDS-PAGE and transferred to PVDF membranes. The blots were probed with primary antibodies, followed by anti-rabbit IgG (Thermo Scientific) secondary antibodies. The primary antibodies against ENL (Cell signaling #14893), AF9 (Novusbio #NB100-1565), DOT1L (Cell signaling #77087), AFF4 (Abcam #ab103586), Cyclin T1 (Cell signaling #81464), H3K79me2 (Cell signaling #5427), H3K79me1 (Cell signaling #9398), Histone H3 (Cell signaling #4499), FLAG (Sigma-Aldrich #F1804), β-Actin (Cell signaling #4970) were used in this study.

Li X., Yao Y., Wu F, & Song Y. (2022). A proteolysis-targeting chimera molecule selectively degrades ENL and inhibits malignant gene expression and tumor growth. Journal of Hematology & Oncology, 15, 41.

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Antibody-based Protein Analysis Protocol

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Antibodies against LDHA (Cell Signaling Technology, Danvers, MA, USA, 3582, 1:2500), DOT1L (Cell Signaling Technology, 77087, 1:1000), H3K79me2 (Cell Signaling Technology, 5427, with 1:1000), BCAT1 (Cell Signaling Technology, Cat.88785, 1:1000), Tubulin (Santa Cruz, Santa Cruz, CA, USA, Cat. sc-5286, 1:2000), Lamin B1 (Cell Signaling Technology, Cat.13435, 1:2000), β-actin (Santa Cruz, Cat. sc-047778, 1:2000), and GAPDH (Santa Cruz, Cat.sc-47724, 1:2000) were used for Western blotting.
Antibodies against LDHA (Cell Signaling Technology, 3582, 1:500), BCAT1 (Cell Signaling Technology, Cat.88785, 1:500), GCLM (Proteintech, Rosemont, IL, USA, Cat.14241, 1:500), and TXNRD1 (Proteintech, Cat.11117, 1:500) were used for immunohistochemistry staining (IHC).
Hydrogen peroxide solution (H₂O₂) (Sigma, St. Louis, MO, USA, Cat.323381), N-acetyl cysteine (NAC) (Sigma, Cat.A7250), DAPI (Sigma, Cat.D9542), sodium 2-hydroxybutyrate (Santa Cruz, Cat. sc-258161), sulfasalazine (SSA) (Sigma, Cat.S0087), and EPZ004777 (Selleck, Washington, DC, USA, Cat.S7353) were commercially obtained.

Li Z., Gu Z., Wang L., Guan Y., Lyu Y., Zhang J., Wang Y., Wang X., Xiong J, & Liu Y. (2023). Nuclear Translocation of LDHA Promotes the Catabolism of BCAAs to Sustain GBM Cell Proliferation through the TxN Antioxidant Pathway. International Journal of Molecular Sciences, 24(11), 9365.

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Immunoblotting Protein Detection Protocol

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For immunoblotting, the protein samples were separated by SDS/PAGE. Subsequently, the protein was transferred to apolyvinylidenedifluoride (PVDF) membrane (Millipore, IBFP0785C). Following, membranes were blocked with 5% no‐fat milk in TBST, and then, the membranes were incubated with specific primary antibodies at 4 °C with a 1 : 1000 dilution of anti‐FOXM1 (Santa Cruz, sc‐376471), or H3K79me2 (Cell Signaling Technology, Beverly, MA, USA, 5427S) overnight. After incubation with the relevant secondary antibodies, the reactive bands were identified using an enhanced chemiluminescence (ECL) detection reagent (Advansta, K‐12045‐D20).

Zhou Z., Chen H., Xie R., Wang H., Li S., Xu Q., Xu N., Cheng Q., Qian Y., Huang R., Shao Z, & Xiang M. (2019). Epigenetically modulated FOXM1 suppresses dendritic cell maturation in pancreatic cancer and colon cancer. Molecular Oncology, 13(4), 873-893.

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Comprehensive Signaling Pathway Analysis

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Cells were harvested in lysis buffer and analyzed by SDS-PAGE with the following antibodies: phospho-AKT (S473), AKT, phospho-S6K (T389), S6K, phospho-S6 (S235/236), S6, phospho-GSK3 (S21), GSK3, phospho-Rb, mme-K, dme-K, tme-K, H3K4-me2, H3K4-me3, H3K9-me2, H3K27-me2, H3K27-me3, H3K36-me2, H3K79-me2 (purchased from Cell Signaling Technology, USA), phospho-PDK1 (S241), PDK1, RAS (bought from Abcam, UK), AMD1 (Proteintech, USA), and Actin (HuaBio, China). Immunoblots were imaged and analyzed using an Odyssey system (LI-COR Biosciences, USA), ImageQuant LAS 4000 (GE Healthcare, USA), and ChemiDoc MP (Biorad, USA).

Xu Y., Fang H., Chen Y., Tang Y., Sun H., Kong Z., Yang F., Kirschner-Schwabe R., Zhu L., Toker A., Xiao N., Zhou B.B, & Li H. (2022). The KRAS-G12D mutation induces metabolic vulnerability in B-cell acute lymphoblastic leukemia. iScience, 25(3), 103881.

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Protein Expression Characterization Protocol

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Cells were harvested and lysed in Laemmli sample buffer (5% SDS, 25% glycerol, 150 mmol/L Tris-HCl pH 6.8, 0.01% bromophenol blue). After protein concentration was measured using BCA protein assay, 0.7 mol/L β-mercaptoethanol was added and protein samples were boiled for 10 minutes. Samples were separated by SDS-PAGE, and protein was transferred onto a polyvinylidene fluoride membrane (Immobilon-P membrane, Millipore). Following antibodies were used: SAE2 (ab58451, abcam), SUMO-2,3 (M114-3, MBL), c-Myc (ab32072, Abcam), GAPDH (sc-20357, Santa Cruz Biotechnology), SUMO-1 (#4930), IRF4 (#4964), CRBN (#71810), cleaved PARP (#5625), Aiolos (#15103), Ikalos (#14859), DOT1L (#77087), H3K79me2 (#5427), PU.1 (#2266), UBC9(#4918) and SAE1(#13585) were from Cell Signaling Technology. Western blot results were visualized using an Odyssey detection system (Licor) or Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).

Du L., Liu W., Pichiorri F, & Rosen S.T. (2022). SUMOylation inhibition enhances multiple myeloma sensitivity to lenalidomide. Cancer Gene Therapy, 30(4), 567-574.

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Cell Proliferation Assay Protocol

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The CellTiter 96 Aqueous ONE Solution Cell Proliferation Assay was purchased from Promega Corp. (Madison, WI, USA). NVP-AUY922 (AUY922) (>99% purity) was from Selleckchem.com (Houston, TX, USA). Ganetespib (STA9090) (98.79% purity) and SNX2112 (98% purity) were purchased from ApexBio (Houston, TX, USA). AT13387 (>98%) was from MedChem Express (Princeton, NJ, USA), CUDC305 (>98% purity) was from AbMole BioScience (Houston, TX, USA), and dimethyl sulfoxide (DMSO) was from Sigma-Aldrich (St. Louis, MO, USA). AUY922, ganetespib, SNX2112, AT13387, and CUDC305 were dissolved in DMSO separately and stored at −20 °C. Polyclonal antibodies against histone H3, H4, H3K4me3, H3K18ac, and H4K12ac were purchased from Abcam (Cambridge, MA, USA). Monoclonal or polyclonal antibodies against acetyl-histone H3, acetyl-histone H4, and H3K27ac were bought from EMD Millipore Corporation (Billerica, MA, USA). Monoclonal or polyclonal antibodies against histone H3K4me1, H3K4me2, H3K23ac, H3K79me1, H3K79me2, H4K8ac, and H4K20me3 were obtained from Cell Signaling Technology (Danvers, MA, USA). Restore Western Blot Stripping Buffer was from Thermo Scientific (Rockford, IL, USA). All other reagents were from Sigma-Aldrich.

Li Q.Q., Hao J.J., Zhang Z., Krane L.S., Hammerich K.H., Sanford T., Trepel J.B., Neckers L, & Agarwal P.K. (2017). Proteomic analysis of proteome and histone post-translational modifications in heat shock protein 90 inhibition-mediated bladder cancer therapeutics. Scientific Reports, 7, 201.

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Adipose Tissue Histone Methylation Profiling

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Histone protein was extracted from adipose tissue treated with Nnmt ASO and control ASO using the EpiQuik Total Histone Extraction Kit from Epigentek. Eight types of histone methylation were measured by western blot analysis using the following antibodies: H3K4me2, H3K9me2, H3K27me2, H3K36me2 and H3K79me2 from Cell Signaling; and H3K4me1, H3K4me3 and H3K9me3 from Abcam. The levels of histone methylation were normalized to total H3 expression. For ChIP, chromatin was first extracted from 3T3-L1 adipocytes treated with or without 10 mM N-methylnicotinamide for 48 h. Immunoprecipitation was performed using EpiQuik Methyl-Histone H3-K4 ChIP kit from Epigentek. Enrichment of methylated H3K4 on Odc and Ssat genes was measured by real-time PCR using EpiTect ChIP qPCR primers from Qiagen (Odc, GPM1030188(+)01A; Ssat, GPM1055920(+)01A). An open reading frame free region (Igx1a) was used as a negative control for ChIP-qPCR. Control IgG showed minimum background among all the regions analysed.

Kraus D., Yang Q., Kong D., Banks A.S., Zhang L., Rodgers J.T., Pirinen E., Pulinilkunnil T.C., Gong F., Wang Y.C., Cen Y., Sauve A.A., Asara J.M., Peroni O.D., Monia B.P., Bhanot S., Alhonen L., Puigserver P, & Kahn B.B. (2014). Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity. Nature, 508(7495), 258-262.

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Total Cellular Protein Extraction and Histone Analysis

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For extraction of total cellular protein, cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, pH 7.4), supplemented with protease inhibitor cocktail (Roche), sodium orthovanadate (1 mM), and PMSF (1 mM). Whole-cell lysates were normalized for protein content by BCA assay (Pierce). Histone extraction was performed using an EpiQuick Total Histone Extraction Kit (Epigentek) and extracted protein was quantified using a Bradford assay.
Proteins were resolved by SDS-PAGE, transferred to a PVDF membrane and probed with the following antibodies: IDH1 (Origene, TA500610), 6×His (Abcam, ab1187), H3K9Me2 (Cell Signaling, 4658p), H3K9Me3 (Abcam, ab8898), H3K27Me3 (Millipore, 07-449), H3K79Me2 (Cell Signaling, 9757p), total H3 (Cell Signaling, 4499p), and Actin. Secondary anti-mouse and anti-rabbit horseradish peroxidase-conjugated IgG antibodies were used for detection.

Elhammali A., Ippolito J.E., Collins L., Crowley J., Marasa J, & Piwnica-Worms D. (2014). A High-Throughput Fluorimetric Assay for 2-Hydroxyglutarate Identifies Zaprinast as a Glutaminase Inhibitor. Cancer discovery, 4(7), 828-839.

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