Anti g9a

Manufactured by Merck Group

Sourced in United Kingdom, United States

Anti-G9A is a laboratory research tool that inhibits the enzymatic activity of the G9a histone methyltransferase. It is designed to be used in experimental settings to study the biological functions of G9a and its role in cellular processes.

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4 protocols using anti g9a

Protein Extraction and Western Blot Analysis

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Mammalian Protein Extraction Reagent (M-PER, Thermo Scientific, IL, USA) was used to harvest total protein for western analyses except for HIF2α, which was harvested using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Triton extraction buffer was used to harvest histones by acid extraction according to the Abcam histone extraction protocol. Protein concentrations were determined using the Bio-Rad Protein Assay Kit (Bio-Rad, CA, USA). For western blot, primary antibodies used were anti-G9A (Sigma-Aldrich, G6919, 1:1000), anti-JMJD1A (Abcam, Cambridge, UK, ab80598, 1:1000), anti-HIF1α and anti-HIF2α (Novus, CO, USA, NB100-479, 1:1000 and NB100-122, 1:1000 respectively), anti-β-actin (Santa Cruz Biotechnology, TX, USA, sc-1616, 1:500), anti-histone H3 (Millipore, Carrigtwohill, Ireland, 06–755, 1:100), and anti-H3K9me1/2/3 (Active Motif, CA, USA, MABI0306, 1:500, MABI0307, 1:1000 and MABI0308, 1:500 respectively).

Ho J.C., Abdullah L.N., Pang Q.Y., Jha S., Chow E.K., Yang H., Kato H., Poellinger L., Ueda J, & Lee K.L. (2017). Inhibition of the H3K9 methyltransferase G9A attenuates oncogenicity and activates the hypoxia signaling pathway. PLoS ONE, 12(11), e0188051.

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Antibody Validation for Chromatin Research

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The antibodies used in this study were: anti-His-tag (D291-3, MBL, Aichi, Japan), anti-Myc-tag (M047-3, MBL), anti-GFP-tag (M048-3, MBL), anti-GLP (D220-3, MBL), anti-Flag-tag (F1804, Sigma-Aldrich, St Louis, MO, USA), anti-G9a (G6919, Sigma-Aldrich), anti-GST-tag (C1303, APPLYGEN, Beijing, China), anti-mCherry-tag (C1329, APPLYGEN, Beijing, China), anti-H3 (ab1791, Abcam, Cambridge, UK), anti-H4 (ab10158, Abcam), anti-H3K9me2 (ab1220, Abcam), anti-H3K27me3 (ab6002, Abcam), anti-H3K9me3 (ab8898, Abcam), anti-H3K79me1 (ab2886, Abcam), anti-H4K20me1 (ab9051, Abcam), anti-H4K20me2 (ab9052, Abcam), anti-MRE11 (ab12159, Abcam), anti-RPA32 (ab2175, Abcam), anti-phospho-Histone H2AX (Ser139) (05–636, EMD Millipore, Billerica, MA, USA), anti-53BP1 (MAB3802, EMD Millipore), anti-GLP (09–078, EMD Millipore), anti-FK2 (04–263, EMD Millipore), anti-53BP1 (NB100–304, Novus Biologicals, Abingdon, UK), anti-GLP (B0422, Novus Biologicals), anti-DNA-PKcs (sc-1552, Santa Cruz Biotechnology), anti-ATR (sc-1887, Santa Cruz Biotechnology), anti-Actin (sc-58673, Santa Cruz Biotechnology), anti-SET8 (C18B7, Cell Signaling Technology, Danvers, MA, USA), anti-ATM (GTX70103, GeneTex), anti-RNF8 (14112-1-AP, Proteintech, Wuhan, Hubei, China) and anti-RNF168 (21393-1-AP, Proteintech).

Lu X., Tang M., Zhu Q., Yang Q., Li Z., Bao Y., Liu G., Hou T., Lv Y., Zhao Y., Wang H., Yang Y., Cheng Z., Wen H., Liu B., Xu X., Gu L, & Zhu W.G. (2019). GLP-catalyzed H4K16me1 promotes 53BP1 recruitment to permit DNA damage repair and cell survival. Nucleic Acids Research, 47(21), 10977-10993.

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Western Blot Analysis of Chromatin Regulators

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Total proteins were separated by electrophoresis on 4–12% gradient PAGE gels (Bio-Rad, #345–0124) and transferred on nitrocellulose membrane (Bio-Rad, #1620115) for western blot. The following antibodies were used for western blot: anti-G9A (Sigma, #HPA050550), anti-SUV39H2 (mix of 1/3 Active Motif, #61449 and 2/3 Proteintech, #11338-AP, (see Supplementary Figure S6), anti-Brg1 (Euromedex, #2SN-2E12-AS), anti-V5 (Life Technologies, #R960–25), anti-Ubiquitin (mAb P4D1, Cell Signalling, #3936), anti-tubulin (Abcam, #ab56676), anti-HP1α (Euromedex, #2HP-2G9-AS), anti-H3 (Abcam, #ab1791), anti-H3K9me2 (Millipore, #07–441), anti-H3K9me3 (Abcam, #ab8898), anti-Rabbit IgG HRP (GE Healthcare, #NA934V), anti-Mouse IgG HRP (Abcam, #ab6808). Western blot signal was acquired with Odyssey Fc system (Licor) and analyzed with the Image Studio software.

Mauger O., Klinck R., Chabot B., Muchardt C., Allemand E, & Batsché E. (2015). Alternative splicing regulates the expression of G9A and SUV39H2 methyltransferases, and dramatically changes SUV39H2 functions. Nucleic Acids Research, 43(3), 1869-1882.

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Immunoprecipitation and Immunoblotting Analysis

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Cells were lysed in lysis buffer [10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40 and protease inhibitor cocktail] for 30 min on ice, followed by centrifugation at 13 000 g for 15 min at 4°C. Whole-cell lysates (WCLs) were subjected to IP by incubation overnight at 4°C with a primary antibody in the presence of protein G magnetic beads (Bio-Rad). After extensive washing with lysis buffer, the bound proteins were eluted by boiling in SDS-PAGE sample buffer, and analyzed by immunoblot assays. Antibodies used for IP and immunoblot assays are the following: anti-methyl lysine (ab23366, Abcam; NB600-824, Novus Biologicals); anti-HIF-1α (sc-10790, Santa Cruz; 610959, BD Biosciences); anti-HIF-2α (A300-286A, Bethyl Laboratories); anti-G9a (G6919, Sigma); anti-GLP (A301-642A, Bethyl Laboratories); p300 (NB500-161, Novus Biologicals); anti-JMJD2C (NBP1-49600, Novus Biologicals); anti-Pontin (12300S, Cell Signaling Technology); anti-Reptin (8959S, Cell Signaling Technology); anti-FLAG (F3165, Sigma); anti-V5 (R96025, Invitrogen); anti-HA (H9658, Sigma); anti-monomethyl and anti-dimethyl HIF-1α K674 antibodies (Novus Biologicals).

Bao L., Chen Y., Lai H.T., Wu S.Y., Wang J.E., Hatanpaa K.J., Raisanen J.M., Fontenot M., Lega B., Chiang C.M., Semenza G.L., Wang Y, & Luo W. (2018). Methylation of hypoxia-inducible factor (HIF)-1α by G9a/GLP inhibits HIF-1 transcriptional activity and cell migration. Nucleic Acids Research, 46(13), 6576-6591.

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