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Suz12

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SUZ12 is a protein that is a component of the Polycomb Repressive Complex 2 (PRC2), which is involved in epigenetic regulation of gene expression. SUZ12 is essential for the histone methyltransferase activity of PRC2.

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Lab products found in correlation

H3k27me3, by Abcam (5 mentions) Trim37, by Abcam (4 mentions) Tls 55 rotor, by Beckman Coulter (2 mentions) 34 mm centrifuge tube, by Beckman Coulter (2 mentions) Mw gf 1000, by Beckman Coulter (2 mentions) Ab101273, by Fortis Life Sciences (2 mentions) Anti flag magnetic beads, by Merck Group (2 mentions) Trueblot ip beads, by Thermo Fisher Scientific (2 mentions) Trim37, by Merck Group (2 mentions) β actin, by Bioworld Technology (2 mentions) Bcl xl, by Cell Signaling Technology (2 mentions) Bcl 2, by Cell Signaling Technology (2 mentions) Vimentin, by Santa Cruz Biotechnology (2 mentions) Snail, by Cell Signaling Technology (2 mentions) Aebp2, by Novus Biologicals (2 mentions) Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions) Antibodies against ezh2, by Abcam (1 mentions) Hiseq 2500, by Illumina (1 mentions) Total h3, by Abcam (1 mentions) Jarid2, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Anti-SUZ12 (10 citations) SUZ12 antibody (3 citations) EZH2 and SUZ12 antibodies (3 citations) EZH2 and SUZ12 (2 citations)

22 protocols using suz12

1

RIP Assay for RNA-Binding Proteins

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RIP assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore Corp., USA). Cell extracts were harvested with RIP lysis buffer and mixed with magnetic beads and antibodies against EZH2, LSD1 and SUZ12 (Abcam, UK). With anti-IgG antibody as control, the co-precipitated RNA was purified and analyzed by qRT-PCR.
Liu J., Li Z., Yu G., Wang T., Qu G, & Wang Y. (2021). LINC01232 Promotes Gastric Cancer Proliferation through Interacting with EZH2 to Inhibit the Transcription of KLF2. Journal of Microbiology and Biotechnology, 31(10), 1358-1365.
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2

ChIP-seq analysis of chromatin modifications

Cited 4 times
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ChIP was performed for SUZ12 (Abcam), EZH2 (Cell Signaling), JARID2 (Cell Signalling), H3K27me3 (Abcam), total H3 (Abcam), and EP300 (Bethyl) as previously described (Kanhere et al. 2012 (link)), with variations described in the Supplemental Methods. Enrichment of specific gene sequences was measured by qPCR (Applied Biosystems). Libraries were sequenced using an Illumina HiSeq 2500 (50 bp single-end). ChIP-seq reads were trimmed to remove adaptors and low-quality bases and aligned with Bowtie 2 (Langmead and Salzberg 2012 (link)). MACS14 (Zhang et al. 2008 (link)) and MAnorm (Shao et al. 2012 (link)) were used to identify sites at which SUZ12 binding was significantly changed by RNase treatment (P < 0.05).
Beltran M., Yates C.M., Skalska L., Dawson M., Reis F.P., Viiri K., Fisher C.L., Sibley C.R., Foster B.M., Bartke T., Ule J, & Jenner R.G. (2016). The interaction of PRC2 with RNA or chromatin is mutually antagonistic. Genome Research, 26(7), 896-907.
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3

Nuclear Protein Complex Analysis via Sucrose Gradient

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Sucrose gradient sedimentation analysis was performed as described39 (link). Briefly, 10–40% gradients were formed by layering 500 µl NEB1 buffer containing 10%, 20%, 30%, or 40% sucrose in a 11 × 34-mm centrifuge tube (Beckman) and allowed to equilibrate at room temperature for 2 h. Gradients were chilled, loaded with 500 µg MCF7 nuclear extract (adjusted to a volume of 150 µl) or 150 µl molecular weight markers (Sigma MW-GF-1000), and centrifuged in a Beckman TLS-55 rotor at 50,000 rpm (214,000×g) for 14 h. Thirty-six fractions of •45 µl were collected. For the markers, 20 µl of each fraction was electrophoresed and Coomassie stained. For the gradient fractions, 20 µl of fractions were analysed by immunoblotting using EZH2 (Cell Signaling Technology), SUZ12 (Abcam ab12073), TRIM37 (Abcam), RNF2 (Abcam ab101273) and BMI1 (Bethyl Laboratories, A301-694A).
Bhatnagar S., Gazin C., Chamberlain L., Ou J., Zhu X., Tushir J.S., Virbasius C.M., Lin L., Zhu L.J., Wajapeyee N, & Green M.R. (2014). TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein. Nature, 516(7529), 116-120.
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4

ASXL1 Interaction Profiling in KBM5 Cells

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ASXL1 mutation-corrected and uncorrected KBM5 cells were lysed using RIPA buffer (Pierce) supplemented with Halt Protease Inhibitor Cocktail (Pierce). Protein concentrations were quantified using BCA Protein Assay KIT (Thermo Scientific), according to the manufacturer's instructions. In each experiment, equal amounts of extracted proteins were separated by SDS-PAGE and blotted using nitrocellulose membrane. Western blots were carried out using the following antibodies: ASXL1 (Novus Biologicals, H00171023-m05), BAP-1 (Santa Cruz, clone 3C11: sc-13576), EZH2 (Active Motif, 39933), Histone H3 lysine 27 trimethyl (Abcam, ab6147), Histone H3 (Abcam, ab1791), SUZ12 (abcam, ab12073) and β-actin (Sigma, A3854) as loading control.
Immunoprecipitation experiments were performed using The Pierce Classic Magnetic IP/Co-IP Kit (Thermo Scientific), according to the manufacturer's instructions. The BAP1 protein fraction was immunoprecipitated using and BAP1 antibody and stained for ASXL1 and BAP1. Anti-ASXL1 and anti-BAP1 antibodies used for immunoprecipitation were the same as the ones used for Western blot experiments as described above.
Valletta S., Dolatshad H., Bartenstein M., Yip B.H., Bello E., Gordon S., Yu Y., Shaw J., Roy S., Scifo L., Schuh A., Pellagatti A., Fulga T.A., Verma A, & Boultwood J. (2015). ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts. Oncotarget, 6(42), 44061-44071.
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5

Chromatin Immunoprecipitation Assay Protocol

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ChIP assays were performed using the EZ-CHIP Kit, according to the manufacturer’s instructions (Millipore, 17–375). Briefly, 1 × 107 cultured NMCMs with each treatment were washed and collected with ice-cold PBS containing protease inhibitor cocktail. Then, the precipitated cells were completely or partially digested by a moderate enzymatic cocktail with non-crosslinking, obtaining chromatin fragments averaging one to a few nucleosomes in length. After diluting with ChIP dilution buffer, the digested chromatin was pre-cleared using protein A/G agarose beads and incubated with H3K27me3 (Abcam), SUZ12 (Abcam) antibodies, or normal mouse IgG at 4°C overnight. The following day, the antibody-chromatin complexes were recovered by incubation with protein A/G agarose beads at 4°C for 2 hours. After washing and purification, the immunoprecipitated DNA was analyzed by real-time PCR. Results were normalized to the input DNA, and normal mouse IgG was used as a negative control. The ChIP primer sequences are listed in the Supplementary Table 2.
Yu J., Yang Y., Xu Z., Lan C., Chen C., Li C., Chen Z., Yu C., Xia X., Liao Q., Jose P.A., Zeng C, & Wu G. (2020). Long Noncoding RNA Ahit Protects Against Cardiac Hypertrophy through SUZ12-Mediated Downregulation of MEF2A. Circulation. Heart failure, 13(1), e006525.
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6

Probing TRIM37, EZH2, and SUZ12 Interactions

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MCF7 nuclear extract (~1000 µg) was incubated with a TRIM37 (Abcam), EZH2 (Cell Signaling Technology) or SUZ12 (Abcam) antibody at 4°C for 24 h in the presence or absence of ethidium bromide (100 µg ml−1). For immunoprecipitations from fractionated nuclear lysate, fractions 20–22 (which were enriched for TRIM37, EZH2 and SUZ12) were pooled and diluted 15-fold. Immune complexes were captured on rabbit or mouse TrueBlot IP beads (eBioScience), washed three times in NEB1 buffer, and eluted by boiling 10 min in 2× SDS sample buffer. Immunoprecipitations from MCF7 cells ectopically expressing FLAG-tagged TRIM37 were performed similarly, except anti-FLAG magnetic beads (Sigma) were used to capture the immune complexes. Immunoprecipitated proteins were analysed by immunoblotting as described above. Input lanes represent 10–25% of extract loaded in the IP lanes.
Bhatnagar S., Gazin C., Chamberlain L., Ou J., Zhu X., Tushir J.S., Virbasius C.M., Lin L., Zhu L.J., Wajapeyee N, & Green M.R. (2014). TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein. Nature, 516(7529), 116-120.
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7

ChIP-qPCR Assay for Histone Modifications

Cited 1 time
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Chromatin immunoprecipitation (ChIP) assay and qPCR analysis were performed as described previously (11 (link)). Sequences of the primers used in these assays are listed in Table 2. Antibodies used for ChIP experiments were those to FLAG (Cell Signaling; catalog no. 2368), H3K27me3 (Millipore; catalog no. 07-449), H3K4me3 (Millipore; catalog no. 17-614), H3K9Ac (Millipore; catalog no. 17-658), H3K27Ac (Millipore; catalog no. 17-683), SUZ12 (Abcam; catalog no. ab12073), and EZH2 (Cell Signaling; catalog no. 5246). All ChIPs shown are representative of at least 2 independent experiments.
Bazot Q., Paschos K, & Allday M.J. (2018). Epstein-Barr Virus (EBV) Latent Protein EBNA3A Directly Targets and Silences the STK39 Gene in B Cells Infected by EBV. Journal of Virology, 92(7), e01918-17.
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8

Probing TRIM37, EZH2, and SUZ12 Interactions

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MCF7 nuclear extract (~1000 µg) was incubated with a TRIM37 (Abcam), EZH2 (Cell Signaling Technology) or SUZ12 (Abcam) antibody at 4°C for 24 h in the presence or absence of ethidium bromide (100 µg ml−1). For immunoprecipitations from fractionated nuclear lysate, fractions 20–22 (which were enriched for TRIM37, EZH2 and SUZ12) were pooled and diluted 15-fold. Immune complexes were captured on rabbit or mouse TrueBlot IP beads (eBioScience), washed three times in NEB1 buffer, and eluted by boiling 10 min in 2× SDS sample buffer. Immunoprecipitations from MCF7 cells ectopically expressing FLAG-tagged TRIM37 were performed similarly, except anti-FLAG magnetic beads (Sigma) were used to capture the immune complexes. Immunoprecipitated proteins were analysed by immunoblotting as described above. Input lanes represent 10–25% of extract loaded in the IP lanes.
Bhatnagar S., Gazin C., Chamberlain L., Ou J., Zhu X., Tushir J.S., Virbasius C.M., Lin L., Zhu L.J., Wajapeyee N, & Green M.R. (2014). TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein. Nature, 516(7529), 116-120.
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9

RNA Immunoprecipitation Assay Protocol

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RNA immunoprecipitation assays were performed as described by Rinn et al. (2007) (link). In brief, cells were fixed with 1% formaldehyde, treated with DNase I, and sonicated using a sonicator (Vibra-Cell VCX130; Sonics & Materials, Inc.). Sonicated samples were immunoprecipitated with protein G–agarose, and antibodies were raised against SMC1, SUZ12, Ezh2, and IgG (Abcam). The precipitated RNA was released, and cDNA was synthesized. After proteinase K treatment, cDNA was precipitated, and Kcnq1ot1 and GAPDH were detected by semiquantitative PCR using the primers listed in Table S5.
Zhang H., Zeitz M.J., Wang H., Niu B., Ge S., Li W., Cui J., Wang G., Qian G., Higgins M.J., Fan X., Hoffman A.R, & Hu J.F. (2014). Long noncoding RNA-mediated intrachromosomal interactions promote imprinting at the Kcnq1 locus. The Journal of Cell Biology, 204(1), 61-75.
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10

Chromatin Immunoprecipitation (ChIP) Protocol

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Cells (3×106/ChIP) were cross-linked with 1% formaldehyde for 10 min. The cross-linked chromatin was sonicated (30sec ON/OFF for 10 min in a Bioruptor®Pico (Diagenode)) or enzymatically digested (EZ-ZymeTM Chromatin Prep kit (Cat#17-375, Millipore) to generate <500 bp DNA fragments. Chromatin supernatant (1%) was collected as the input before adding the following immunoprecipitating antibodies: H3K4me3 (Diagenode C15410003), H3K27me3 (Diagenode C15410069 or Abcam ab6002), H3K27ac (Diagenode C15410174), H3K4me1 (Diagenode C15410037) and SUZ-12 (Abcam ab12073). Magnetic Dynabeads Protein A (Invitrogen) or protein G Agarose (EZ-ChIPTM Chromatin Immunoprecipitation kit (Cat#17-371, Millipore)) were used to bind the antibody/antigen/DNA complex. ChIP samples were washed, crosslinks were reversed and ChIP DNA was isolated as the template for real-time qPCR using either SYBR Green (BioRad) or custom designed TaqMan assays (Tables S5 and S6).
Kuo F.C., Neville M.J., Sabaratnam R., Wesolowska-Andersen A., Phillips D., Wittemans L.B., van Dam A.D., Loh N.Y., Todorčević M., Denton N., Kentistou K.A., Joshi P.K., Christodoulides C., Langenberg C., Collas P., Karpe F, & Pinnick K.E. (2022). HOTAIR interacts with PRC2 complex regulating the regional preadipocyte transcriptome and human fat distribution. Cell Reports, 40(4), 111136.
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