To confirm the efficiency of subcellular fractionation and EDTA treatment to dissociate ribosomes, EDTA‐treated and untreated subcellular fractions from MKRN1 knockdown and shGFP control ESC clones were subjected to immunoblot analysis with the following primary antibodies: rabbit anti‐calnexin (1:1,000; Enzo Life Sciences), rabbit anti‐PABP (1:1,000; Cell Signaling Technologies), rabbit anti‐MOV10 (1:1,000; Bethyl Laboratories), anti‐histone H3K27‐me3 (1:1,000; Abcam), and rabbit anti‐MKRN1 antibody (1:1,000; Abcam). Immunoblot analysis verified that the ER luminal protein marker, calnexin, was uniquely detected in ER‐enriched fractions and not visible in soluble (cytoplasmic). Moreover, immunoblotting for the nuclear marker, H3K27‐me3, confirmed that nuclear proteins were absent from the ER‐enriched and soluble fractions. The effectiveness of EDTA treatment to dissociate mRNPs from membrane‐bound ribosome was gauged by immunoblotting for mRNP protein markers: PABP and the RNA helicase MOV10. Immunoblotting for MKRN1 confirmed its depletion exclusively in MKRN1 knockdown ESC lysates, and its subcellular localization in undifferentiated ESCs represented by shGFP control lysates.
Anti histone h3k27me3
Manufactured by Abcam
Anti-histone H3K27me3 is a laboratory reagent used for the detection and measurement of histone H3 trimethylated at lysine 27 (H3K27me3). This epigenetic mark is associated with transcriptional repression. The product can be used in various applications, such as Western blotting, immunohistochemistry, and chromatin immunoprecipitation.
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Lab products found in correlation
Anti histone h3k4me3, by Abcam (3 mentions)
Anti histone h3k27ac, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Rabbit anti calnexin, by Enzo Life Sciences (1 mentions)
Miseq next generation sequencer, by Illumina (1 mentions)
Thruplex dna seq kit, by Takara Bio (1 mentions)
Chip grade protein g magnetic beads, by Cell Signaling Technology (1 mentions)
Anti runx2, by Cell Signaling Technology (1 mentions)
Anti histone h3k27ac, by Active Motif (1 mentions)
Anti runx3, by Cell Signaling Technology (1 mentions)
Anti flag, by Merck Group (1 mentions)
S220 sonicator, by Covaris (1 mentions)
Ab8898, by Abcam (1 mentions)
Ab1220, by Abcam (1 mentions)
Ab6002, by Abcam (1 mentions)
Sc 899, by Santa Cruz Biotechnology (1 mentions)
Anti ezh2, by Merck Group (1 mentions)
Anti jarid2, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
Anti suz12, by Merck Group (1 mentions)
7 protocols using anti histone h3k27me3
1
Validating Ribosome Dissociation and Subcellular Fractionation
2
ChIP-Seq of Mesenchymal Chondrosarcoma Cells
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ChIP-Seq was carried out using the method previously described using a biological duplicate (58 (link)). Briefly, 5 × 106 mesenchymal chondrosarcoma cells per immunoprecipitation were cross-linked with 1% formaldehyde for 10 minutes at room temperature. Chromatin was sheared in SDS lysis buffer containing 1% SDS, 10 mM EDTA, and 50 mM Tris, pH 8.0, to an average size of 400–500 bp using a Covaris S220 sonicator for 15 minutes. ChIP was carried out with 5 μg anti-FLAG (Sigma-Aldrich), anti-histone H3K27ac (Active Motif), anti-histone H3K27Me3 (Abcam), anti-histone H3K4Me3 (Abcam), anti-Runx2 (Cell Signaling), or anti-Runx3 (Cell Signaling) antibodies. The antibody-bound protein/DNA complexes were immunoprecipitated using ChIP grade protein G magnetic beads (Cell Signaling).
Immunoprecipitated DNA was then purified and subjected to secondary sonication to an average size of 150–350 bp. Libraries were prepared according to instructions accompanying the ThruPLEX DNA-Seq kit (Rubicon Genomics). The ChIP DNA was end modified and adapters were ligated. DNA was PCR amplified with Illumina primers, and Illumina-compatible indexes were added. The library fragments of approximately 300-500 bp were band-isolated from an agarose gel. The purified DNA was sequenced on an Illumina MiSeq next-generation sequencer following the manufacturer protocols.
Immunoprecipitated DNA was then purified and subjected to secondary sonication to an average size of 150–350 bp. Libraries were prepared according to instructions accompanying the ThruPLEX DNA-Seq kit (Rubicon Genomics). The ChIP DNA was end modified and adapters were ligated. DNA was PCR amplified with Illumina primers, and Illumina-compatible indexes were added. The library fragments of approximately 300-500 bp were band-isolated from an agarose gel. The purified DNA was sequenced on an Illumina MiSeq next-generation sequencer following the manufacturer protocols.
3
Chromatin Immunoprecipitation of HIV-1 Genome
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ChIP was performed as previously described (24 (link)), with the Pierce agarose ChIP kit (Thermo Scientific). For ChIP, anti-RNAP II (17-672 [Millipore] or sc-899 [Santa Cruz]), anti-EZH2 (17-662; Millipore), anti-EED (17-10034; Millipore), anti-SUZ12 (17-661; Millipore), anti-Jarid2 (AB192252; Abcam, Inc.), anti-histone H3 (AB1791; Abcam, Inc.), anti-histone H3K27me3 (AB6002; Abcam, Inc.), anti-EHMT2 (3356S; Cell Signaling), anti-SUV39H1 (8729S; Cell Signaling), anti-KDM1 (LSD1) (2139S; Cell Signaling), anti-trimethyl histone H3 (Lys9) (AB8898, Abcam, Inc.), and anti-dimethyl histone H3 (Lys9) (AB1220; Abcam, Inc.) antibodies were used. The percentage-of-input method was used to calculate the enrichment of proteins in specific regions of the HIV-1 genome.
4
Western Blot Analysis of Epigenetic Markers
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Cells were lysed with lysis buffer (100 mM Tris–HCl, pH 6.8, 100 mM DTT, 1% SDS, 10% glycerol). Proteins were separated by 10–12% SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk in phosphate-buffered saline (PBS) for 1 h before incubation with primary antibody overnight at 4 °C. Membranes were washed and blocked with 5% milk and incubated with different primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies. The primary antibodies used in western blotting included anti-Fos B(F-7) (sc-398595; Santa Cruz; 1:1000 dilution), anti-CEBPB (sc-7962; Santa Cruz; 1:1000 dilution), anti- histone H3K27me3 (ab6002, Abcam; 1:1000 dilution), anti-histone H3K27ac (#8173, Cell Signaling; 1:1000 dilution) and anti-GAPDH (sc-47724; Santa Cruz; 1:5000).
5
Protein Extraction and Western Blot Analysis
6
HeLa Cell Line Cloning and Maintenance
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All WT and TAF-I KD HeLa cell lines were cloned by picking up well-isolated drug-resistant colonies using cloning rings33 (link). TAF-I KD HeLa cell lines, HEK293T cells, T98G cells, A549 cells, and HCT116 cells were maintained in Dulbecco modified Eagle medium (DMEM, Nissui) containing 10% FBS. Antibodies used in this study were as follows: anti-TAF-Iα/β antibody (monoclonal antibody KM1720; Kirin-Kyowa Hakko), anti-β-actin, and anti-Sp1 antibodies from SIGMA; anti-histone H3, anti-histone H3 K4me3, anti-histone H3 K27me3, anti-pol II (8WG16), anti-histone H1.2, anti-histone H1.5, and anti-histone H1X antibodies from Abcam; anti-c-Myc antibody from CST; normal rabbit IgG, and anti-histone H3 K9K14ac antibodies from Millipore; anti-histone H1.0 antibody from ProteinTech Group, Inc. Actionomycin D was purchased from SIGMA. Decitabine was purchased from BioVision.
7
Western Blot Analysis of Transcription Factors
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Cells were lysed with lysis buffer (100 mM Tris-HCl, pH 6.8, 100 mM DTT, 1% SDS, 10 % glycerol). Proteins were separated by 10-12% SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk in phosphate-buffered saline (PBS) for 1 h before incubation with primary antibody overnight at 4 °C. Membranes were washed and blocked with 5% milk and incubated with different primary antibodies overnight at 4℃, followed by incubation with secondary antibodies. The primary antibodies used in western blotting included anti-Fos B(F-7) (sc-398595; Santa Cruz; 1:1000 dilution), anti-CEBPB (sc-7962; Santa Cruz; 1:1000 dilution), anti-histone H3K27me3 (ab6002, Abcam; 1:1000 dilution), anti-histone H3K27ac (#8173, Cell Signaling; 1:1000 dilution) and anti-GAPDH (sc-47724; Santa Cruz; 1:5000).
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