Tumor cells (2 × 106) were prepared for chromatin immunoprecipitation (ChIP) assay with the ChIP assay kit (Millipore Technology, Billerica, MA) according to the manufacturer’s protocol. The resulting precipitated DNA samples were analyzed using quantitative real-time PCR to amplify a 408-bp region of the MOB1 promoter with the primers 5′-CGC TTA TAG AGG TCC CTG CAT AAC-3′ (forward) and 5′-TCA GGA TTC GGA GCT GGC TAG-3′ (reverse); a 80-bp region of the YAP promoter with the primers 5′-TCG CCG CTT CTC CAC CT-3′(forward) and 5′- GAC GCG CAC CCC CTG AC-3′ (reverse); a 101-bp region of the TAZ promoter with the primers 5′- AGA AAC CTC AGA GTG ACT AAA AT − 3′(forward) and 5′- TAA GAG TCA GAT GAG CAG AGA TG − 3′ (reverse). The antibodies used in the ChIP assay were anti-KDM2B (#09–864, Millipore), anti-H3K27me3 (#9733, Cell Signaling Technology), anti-H3K36me2 (#2901, Cell Signaling Technology), anti-H3K4me3 (#9751, Cell Signaling Technology). The experiments were performed in triplicate and repeated twice.
Anti h3k36me2
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-H3K36me2 is a laboratory reagent that specifically recognizes and binds to the dimethylated form of histone H3 at lysine 36. This modification is associated with active transcription and is commonly used as a marker for this chromatin state.
Automatically generated - may contain errors
Lab products found in correlation
Anti h3k27me3, by Cell Signaling Technology (4 mentions)
Anti h3k4me2, by Cell Signaling Technology (4 mentions)
Anti h3k79me2, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Anti h3k27me2, by Cell Signaling Technology (3 mentions)
Anti histone h3, by Cell Signaling Technology (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Mabe191, by Abcam (2 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibodies, by GE Healthcare (2 mentions)
Anti h3k36me3, by Active Motif (2 mentions)
Anti his, by ZSGB-BIO (2 mentions)
Anti β tubulin, by Cell Signaling Technology (2 mentions)
Anti vinculin, by Cell Signaling Technology (2 mentions)
Anti lamin b1, by Cell Signaling Technology (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti ha, by BioLegend (2 mentions)
Anti dot1l, by Cell Signaling Technology (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
9 protocols using anti h3k36me2
1
Chromatin Immunoprecipitation Analysis of Tumor Cell Epigenetics
2
Histone Modification Analysis Protocol
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The following antibodies were used in this study: anti-KDM2A (12 (link)), anti-KDM2B (15 (link)), anti-BRG1 (EPNCIR111A, Abcam), anti-H3, anti-H3K36me1, anti-H3K36me2 (12 (link)), anti-H3K36me3, anti-H4 (L64C1, Cell Signalling), anti-H2AK119ub1 (D27C4, Cell Signalling), anti-H3K27me3 (45 (link)), anti-Rbp1-NTD (D8L4Y, Cell Signalling), anti-Rbp1-CTD-Ser5P (D9N5I, Cell Signalling), anti-Rbp1-CTD-Ser2P (E1Z3G, Cell Signalling). Anti-H3 and anti-H3K36me antibodies were prepared in-house by rabbit immunization with synthetic peptides (PTU/BS Scottish National Blood Transfusion Service) and antibodies were purified on peptide affinity columns.
3
Western Blot Analysis of Histone Modifications
4
Western Blot Analysis of Protein Expression
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Total cell lysates were prepared in 2× sample loading buffer [250 mM Tris-HCl (pH 6.8), 10% glycerol, 4% sodium dodecyl sulfate (SDS), 2% β-mercaptoethanol, 0.006% bromophenol blue, 5 mM sodium orthovanadate, and 50 mM sodium fluoride; Bio-Rad, Hercules, CA, USA]. The protein concentrations of samples were quantified using the bicinchoninic acid (BCA) method [34 (link)] and a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (5–20 μg) were separated by 6–13% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich) and then probed with anti-DOT1L, anti-H3K79me2, anti-β-Actin, anti-histone H3, anti-H3K4me2, anti-H3K27me2, anti-H3K36me2, anti-H3K79me1, anti-H3K79me3, and anti-Vimentin antibodies purchased from Cell Signaling Technology (Beverly, MA, USA) or with anti-E-cadherin and anti-N-cadherin antibodies purchased from (BD Biosciences, San Jose, CA, USA). The blots were detected using a WEST-Queen detection system (iNtRON Biotechnology, Seongnam, Korea) [35 (link)].
5
Western Blot Analysis of WHSC1 and Epigenetic Markers
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After treating the OCCC cells with WHSC1-specific siRNAs or GSK126 for the indicated times at the indicated concentrations, total protein was extracted and transferred to a nitrocellulose membrane as previously described [16 (link), 17 ]. Primary antibody diluted with blocking buffer was added to the membrane and reacted overnight at 4 °C. The primary antibodies used in this study were anti-WHSC1 (75,359, Abcam, Cambridge, UK), anti-EZH2 (PA0575, Leica Biosystems, Wetzlar, Germany), anti-H3K36me2 (2901, Cell Signaling Technology, Danvers, MA, USA), anti-H3K27me3 (9733, Cell Signaling Technology), and anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA).
6
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Total cell lysates were prepared in 2× sample loading buffer (250 mM Tris-HCl [pH 6.8], 10% glycerol, 4% SDS, 2% β-mercaptoethanol, 0.006% bromophenol blue, 5 mM sodium orthovanadate, and 50 mM sodium fluoride; Bio-Rad). The protein concentrations of samples were measured using the bicinchoninic acid method.40 (link) Equal amounts of protein (520 μg) were separated by 8%–15% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA).41 (link) The membranes were blocked with 5% BSA (Sigma-Aldrich) and then probed with anti-E-cadherin, anti-N-cadherin (BD Biosciences), anti-DOT1L, anti-H3K4me2, anti-H3K27me2, anti-H3K36me2, anti-H3K79me2, anti-Histone H3 (Cell Signaling Technology, Beverly, MA, USA), anti-ZEB1, anti-vimentin, or anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) antibodies. The blots were detected with an enhanced chemiluminescence (ECL) detection kit (GE Healthcare, Little Chalfont, UK).
7
Protein Extraction and Western Blot Analysis
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Total cell lysates were prepared in 2× sample loading buffer [250 mM Tris-HCl (pH 6.8), 10% glycerol, 4% sodium dodecyl sulfate (SDS), 2% β-mercaptoethanol, 0.006% bromophenol blue, 5 mM sodium orthovanadate, and 50 mM sodium fluoride; Bio-Rad, Hercules, CA, USA]. The protein concentrations of samples were quantified using the bicinchoninic acid (BCA) method [38 (link)] and a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein (5–20 μg) were separated by 6–13% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich) and then probed with anti-DOT1L, anti-H3K79me2, anti-β-Actin, anti-histone H3, anti-H3K4me2, anti-H3K9me2, anti-H3K27me2, anti-H3K36me2, and anti-Vimentin antibodies purchased from Cell Signaling Technology (Beverly, MA, USA) or with anti-E-cadherin and anti-N-cadherin antibodies purchased from BD Biosciences (San Jose, CA, USA). The blots were detected using a WEST-Queen detection system (iNtRON Biotechnology, Seongnam, Republic of Korea).
8
Histone Modification Profiling Assay
9
Western Blot Analysis of Histone Modifications
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Fractionated or whole cell lysates were resolved by SDS-PAGE, transferred to a nitrocellulose or PVDF membrane, blocked in 5% non-fat milk in PBS plus 0.5% Tween-20, probed with primary antibodies, and detected with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (GE Healthcare). Primary antibodies were: anti-H3K36me2 (Cell Signaling Tech, 2901), anti-H3K36me3 (Active Motif, 61101), anti-NSD1 (Abbexa, abx135901), anti-NSD2 (Millipore Sigma, MABE191), anti-SETD2 (Abcam, ab31358), anti-DNMT3A (Abcam, ab188470), anti-Vinculin (Cell Signaling Tech, 13901), anti-His (ZSGB-Bio,TA-02), anti-Lamin B1 (Cell Signaling Tech, 12586), anti-β-Tubulin (Cell Signaling Tech, 2128), anti-β-actin (Abcam, ab8224), anti-H3 (Abcam, ab1791) and anti-HA (Biolegend, 901501). The specificities of anti-H3K36me3 and anti-H3K36me2 antibodies were validated using Setd2 knockout and Nsd1/2 double knockout cell lines.
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