RIP was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). The RNAs were immunoprecipitated using anti-EZH2 (Cell Signaling Technology) antibody. Final analysis was performed using qRT-PCR and shown as fold enrichment of HOTAIR. The RIP RNA fraction Ct value was normalized to the input RNA fraction Ct value. ChIP was performed using the EZ ChIP Chromatin Immunoprecipitation Kit (Millipore), according to the manufacturer’s protocol. Briefly, cross-linked chromatin was sonicated into 200-1,000-bp fragments. The chromatin was immunoprecipitated by using anti-EZH2 (Cell Signaling Technology) and anti-H3K27me3 (Millipore) antibodies. qRT-PCR was conducted to detect the relative enrichment of the miR-218-2 promoter. Primers are listed in Table S2 .
Anti ezh2
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-EZH2 is a lab equipment product offered by Cell Signaling Technology. It is an antibody that specifically targets the EZH2 protein, which plays a role in epigenetic gene regulation.
Automatically generated - may contain errors
Lab products found in correlation
Anti h3k27me3, by Cell Signaling Technology (32 mentions)
Anti h3k27me3, by Merck Group (17 mentions)
Anti β actin, by Merck Group (14 mentions)
Anti gapdh, by Cell Signaling Technology (13 mentions)
Anti suz12, by Cell Signaling Technology (9 mentions)
Anti stat3, by Cell Signaling Technology (8 mentions)
Anti ki67, by Cell Signaling Technology (8 mentions)
Anti h3, by Cell Signaling Technology (6 mentions)
Anti β actin, by Cell Signaling Technology (6 mentions)
Anti p stat3, by Cell Signaling Technology (6 mentions)
Anti e cadherin, by Cell Signaling Technology (6 mentions)
Anti gapdh, by Merck Group (6 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (5 mentions)
Ez chip chromatin immunoprecipitation kit, by Merck Group (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Anti h3k27me3, by Diagenode (4 mentions)
Anti h3, by Abcam (4 mentions)
Quantity one software, by Bio-Rad (4 mentions)
Anti arid1a, by Santa Cruz Biotechnology (4 mentions)
Anti β actin, by Santa Cruz Biotechnology (4 mentions)
149 protocols using anti ezh2
1
EZH2 Regulation of HOTAIR and miR-218-2
2
Comprehensive Antibody Panel for Cellular Analyses
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The following antibodies were purchased: for Western blotting, anti-β-catenin (1:5000; Abcam, cat. no. ab32572), anti-β-actin (1:5000; Bioss, cat. no. bs-0061R), anti-CDK5RAP2 (1:2000; Abcam, cat. no. ab70213), anti-survivin (1:2000; Abclonol, cat. no. A1551), anti-ALDH1 (1:1000; Cell Signaling Technology, cat. no. 54135), anti-Notch1 (1:1000, Abcam, cat. no. ab52627), anti-EZH2 (1:1000; Cell Signaling Technology, cat. no. 5246), anti-CCND1 (1:1000, Cell Signaling Technology, cat. no. 55506), and horseradish peroxidase-conjugated secondary antibodies (1:1000; Cell Signaling Technology, Inc.); for IHC analyses, anti-CDK5RAP2 (1:400; Abcam, cat. no. ab235893), anti-ALDH1 (1:50; Abcam, cat. no. ab52492), anti-SOX2 (1:100; Abcam, cat. no. Ab92494), anti-CD44 (1:4000; Abcam, cat. no. ab189524), anti-CD133 (1:1000; Abcam, cat. no. ab222782), anti-Notch1 (1:150; Abcam, cat. no. ab52627), anti-EZH2 (1:200; Cell Signaling Technology, cat. no. 5246), and anti-CCND1 (1:200; ABclonal, cat. no. A19038); and for immunofluorescence analyses, anti-α-tubulin (1:500, YL1/2; Santa Cruz Biotechnology, cat. no. sc-53029), anti-γ-tubulin (1:1000, GTU88; Sigma-Aldrich, cat. no. T5326), and Alexa Fluor-conjugated secondary antibodies (1:500; Thermo Fisher Scientific).
3
Quantitative Analysis of Chromatin Modifications
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Cells were lysed in ice-cold lysis buffer (20 mM Tris–HCl pH 8, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 0.5% Triton X-100 + protease inhibitors) for 15 min at 4 °C. Samples were then sonicated in a Q800 Waterbath sonicator (QSonica) for 15 cycles of 8 s ON, 15 s OFF. Lysates were cleared after 15 min centrifugation at 10,000 g, and protein concentration was determined using Pierce BCA Protein assay kit (Thermo Fisher). Protein extracts were resolved by SDS-PAGE, blotted to PVDF membranes and probed using the following antibodies: anti-EZH2 (Cell Signaling Technologies, Cat nb#5246, 1:1000), anti-H3K27me3 (Diagenode, Cat nb# C15200181-50, 1:5000), anti-H3K27me2 (Abcam, Cat nb# ab24684, 1:1000), anti-H3K27me1 (Active Motif, Cat nb# 61015, 1:1000), anti-H3 (Abcam, Cat nb# ab1791, 1:2000), GAPDH (Millipore, Cat nb# MAB274, 1:10,000), actin (Abcam, Cat nb# ab3280, 1:5000). Signal was detected using IRDye 680RD and 800CW secondary antibodies following the manufacturer’s instructions (LI-COR). Membranes were acquired on an Odyssey Imager (LI-COR) and quantified using Image Studio (LI-COR). Uncropped images of all western blots are provided in the Source Data file.
4
Chromatin Immunoprecipitation for Epigenetic Profiling
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Chromatin immunoprecipitation (ChIP) assays were conducted using an EZ-ChIP kit (Millipore) with anti-EZH2 (Cell Signaling Technology), anti-H3K27me3 (Millipore) or anti-IgG (Millipore) antibodies. The precipitated DNA was analysed by qRT-PCR.
5
Chromatin Immunoprecipitation for Epigenomic Analysis
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Chromatin immunoprecipitation was performed as described previously [8 (link)]. In brief, 1 × 107 cells were crosslinked with 1% formaldehyde, lysed by lysis buffer, and chromatin in the lysate was fragmented into 100–500 bps by sonication. Protein-DNA complexes were immunoprecipitated (IP) by 2 μg antibodies coupled with Dynal magnetic beads (Invitrogen). ChIP-grade antibodies used were anti-AR (Millipore; 06-680), anti-EZH2 (Cell signaling; #4905), anti-H3K27me3 (Diagenode; C15200181-50) and ant-IgG (Abcam; ab2410). The IP or input DNA was subjected to elution, reverse crosslink and purification. Purified IP and input DNA were analyzed by PCR quantification using SYBR® Premix Ex Taq™ Kit (TaKaRa). The primer sequences for individual genes were listed in the supplementary information. Promoter enrichment of EZH2, AXIN2, NKD1, PPP2R2B, PRICKLE1 and SFRP5 conjugated with respective proteins was determined and shown as the percentage of input DNA. IgG antibody was used as a negative control.
6
Western Blotting for Protein Profiling
7
Quantitative Western Blot Analysis
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Cell protein lysates were separated by 10% sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, transferred to 0.22 μm NC membranes (Sigma) and incubated with specific antibodies. The ECL chromogenic substrate was quantified by densitometry (Quantity One software; Bio-Rad). An anti-GAPDH antibody was used as a control, anti-EZH2, and anti-Bcl-xl antibodies (1:1000) were purchased from Cell Signaling Technology, Inc., and anti-IL24 antibodies (1:1000) were purchased from Abcam.
8
UCA1 RNA-Protein Pull-Down Assay
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RNA pull-down assay was performed using Magnetic RNA-Protein Pull-Down Kit (Pierce, USA) according to the manufacturer's instructions. First, the full length of UCA1 was synthesized using RiboMAX™ Large Scale RNA Production Systems (Promega, USA). After biotin labeling, UCA1 was bound to the beads for protein binding. Cell protein lysate was added with RNA-bound beads for immunoprecipitation. Beads were washed three times and boiled in SDS buffer, and the retrieved protein was detected by western blot analysis. The antibody for RNA pull-down assay was anti-EZH2 (1:1000, Cell Signaling Technology, USA).
9
Protein Expression Analysis in PDX Tumors
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Lysates were obtained from frozen PDX tumors collected immediately and after 15 days from the end of treatment with EPZ-011989 as well as from the PDX-derived cell line after different intervals of exposure to EPZ-011989, alone or in association with Baf1A, or following transfection with siATG5 or siHMGA2. Lysates from ES-1 cells exposed to doxorubicin, 4-hydroperoxycyclophosphamide, and gemcitabine were also collected. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes and incubated with primary monoclonal antibodies anti-EZH2 (#5246, Cell Signaling), anti-H3K27me3 (#9733, Cell Signaling), anti-H3 acetyl K27 (H3K27ac, #8173, Cell Signaling), anti-EGR1 (#4153, Cell Signaling), anti-LC3B (#2775 Cell Signaling), anti-HMGA2 (#5269, Cell Signaling), anti-cleaved CCP32 (#9661, Cell Signaling), anti-cleaved PARP (#9541, Cell Signaling), anti-ATG5 (A0856, Sigma, St. Louis, MO, USA), anti-β-tubulin (T5201, Sigma, St. Louis, MO, USA), anti-β-actin (A2066, Sigma, St. Louis, MO, USA), and anti-vinculin (V9131, Sigma, St. Louis, MO, USA).
10
ChIP Assay for PRC2 Targeting
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Chromatin immunoprecipitation (ChIP) assay was performed to evaluate PRC2 targeting and H3K27me3 levels at specific promoters as described70 (link),71 (link). Briefly, minced hearts or NRVMs were fixed with 1% formaldehyde for 10 min at room temperature, and then quenched by 125 mM glycine. The samples were homogenized in lysis buffer containing 20 mM Tris-HCl (pH8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1× complete protease inhibitor tablet, and sonicated to generate chromatin samples with averaged fragment sizes of 200-1000 bp. After pre-cleared with Protein A sepharose beads, samples were incubated with anti-H3K27me3 antibody (Cell Signaling Technologies, MA, USA), anti-Ezh2 (Cell Signaling Technologies (#5246) for ChIP in mouse hearts ; Santa Cruz Biotechnology (#292275) for ChIP in NRVMs) or normal control IgG at 4°C over night on an inverse rotator. The antibody-chromatin complexes were then pelleted with BSA/Salmon sperm DNA blocked Protein A sepharose beads. After standard washes, the immunoprecipitated DNA was eluted and purified with PCR purification kit (Qiagen). RT-PCR was then performed using primers targeting the promoter regions of hypertrophy-related genes as listed in supplementary Table 5 .
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