HEK (human embryonic kidney)-293T cells were purchased from the A.T.C.C. (Manassas, VA, U.S.A.) and maintained in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (Gibco) supplemented with 10 % FBS. Cells (5 × 105) were seeded in each well of a six-well plate in 2 ml of medium. Inhibitors were added to each well at a final concentration of 0.2, 1, 5, 10 or 15 μM in the presence of 1 % DMSO. For the control sample only 1 % DMSO was added to the well. Cells were trypsinized after 48 h and deposited on to microscope slides by cytospin (200 rev./min for 10 min). For GFP visualization, cells were transfected using Lipofectamine™ 2000 according to the manufacturer’s protocol (Invitrogen). Transfected HEK-293T cells were FACS-sorted on the basis of GFP intensity (BD FACSAria II). Cytospinning, fixation and immunofluorescence were performed as described previously [27 (link)]. The primary antibodies used for immunofluorescence include anti-H3K9me3 (catalogue number 07-523; Millipore), anti-H4K20me3 (histone H4 trimethylated at Lys20; catalogue number 39180; Active Motif) and anti-HP1γ (catalogue number MAB3450; Millipore). Images were collected on a Zeiss Axiovert 200 imaging system equipped with an Axiocam MR digital camera controlled by AxioVision software or on a Zeiss LSM Pascal confocal microscope.
Anti h4k20me3
Manufactured by Active Motif
Sourced in United States
Anti-H4K20me3 is a laboratory product that detects the trimethylation of histone H4 at lysine 20. This histone modification is involved in various cellular processes, including DNA repair and chromatin regulation.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Axiocam mr digital camera, by Zeiss (1 mentions)
Lsm pascal confocal microscope, by Zeiss (1 mentions)
Anti h3k9me3, by Merck Group (1 mentions)
Anti hp1γ, by Merck Group (1 mentions)
Axiovert 200 imaging system, by Zeiss (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Facsaria 2, by BD (1 mentions)
Anti satb2, by Abcam (1 mentions)
Anti h3k9me2, by Active Motif (1 mentions)
Dynamo flash supermix, by Thermo Fisher Scientific (1 mentions)
Cfx384 touch pcr system, by Bio-Rad (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Anti h3k4me1, by Abcam (1 mentions)
Anti h3k4me2, by Abcam (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Immun star westernc kit, by Bio-Rad (1 mentions)
Anti ccnd2, by Cell Signaling Technology (1 mentions)
Anti smyd3, by Abcam (1 mentions)
4 protocols using anti h4k20me3
1
Investigating Epigenetic Changes in HEK-293T Cells
2
Chromatin Immunoprecipitation from Cortical Tissue
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We thawed the cortices derived from a single brain and filtered the tissues into a single-cell suspension using gentle mechanical dissociation. We washed cells twice with PBS containing protease inhibitor cocktail (Cat# 78437; Thermo Scientific) and re-suspended them in medium (DMEM F-12 Cat# 11320-033; Invitrogen) containing 0.1 volume crosslinking solution [33 (link)]. We carried out chromatin immunoprecipitation reactions following the previously published protocol [34 (link)]. The specific antibodies used in this study include anti-H3K9me2 (Cat# 39239; Active Motif, RRID:AB_2793199), antiH3K9me3 (Cat# 05-1242; Millipore-Sigma, RRID:AB_1587136), antiH4K20me3 (Cat# 91107; Active Motif, RRID:AB_2793777), anti-SATB2 (Cat# ab34735; Abcam, RRID:AB_2301417) and the negative IgG control (Cat# SC-2027; Santa Cruz, RRID:AB_737197). We used antibodies for modified histones and the IgG control at a concentration of 1 µg/ChIP reaction and 4 µg/ChIP reaction for SATB2. We purified precipitated DNA using the QIAquick PCR Purification Kit (catalog # 28106, Qiagen) and assayed the enrichment of the indicated sequences using quantitative PCR. We performed qPCR using the Dynamo Flash supermix (Cat# F-415XL; Thermo Scientific) on a Bio-Rad CFX384 Touch PCR system. Primer sequences are listed in Additional file 1 .
3
Western Blot Analysis of Epigenetic Markers
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Cells were collected with a gum rubber-scraping device, lysed with RIPA buffer (sc-24948, Santa Cruz Biotechnology Inc.) and protein concentration was determined using BCA assay (Thermo Scientific, Waltham, MA, USA) according to manufacturer's information. Subsequently, 30 μg of total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes and incubated with antibodies against anti-SMYD3 (dilution: 1:500, Abcam, Cambridge, UK), against anti-CCND2 (dilution: 1:500, Cell Signaling, Danvers, MA, USA), anti-H3K4me1 (dilution: 1:1000, Abcam), anti-H3K4me2 (dilution: 1:1000, Abcam), anti-H3K4me3 (dilution: 1:400 Abcam), anti-H3K27me3 (dilution: 1:500 Millipore, Billerica, MA, USA), anti-H4K20me3 (dilution: 1:500 Active Motif, Carlsbad, CA, USA) and as input control anti-histone H3 rabbit antibody (dilution: 1:500, Abcam), anti-histone H4 rabbit antibody (dilution: 1:500, Abcam) and β-actin (dilution: 1:8000, Sigma-Aldrich, Schnelldorf, Germany), when appropriate. The blots were developed using Immun-Star™ WesternC™ Kit according to manufacturer's indications (BioRad, Hercules, CA, USA). All the experiments were performed in triplicate. Relative optical density determination was performed using QuantityOne® Software version 4.6.6. (Biorad) and proteins levels were normalized using β-actin levels as reference.
4
Protein Extraction and Western Blotting
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Whole cell lysates were collected and quantified with a Micro BCA Protein Assay kit (Thermo Scientific). Nuclear extracts were collected with EpiQuick Nuclear Extraction kit (Epigentek, Farmingdale, NY), then quantified. Lysates or nuclear extracts were subjected to SDS-PAGE and western immunoblotting (WB) as described before 28 (link). The following antibodies were used: Anti-Mof (A300-992A) from Bethyl Laboratories (Montgomery, TX); anti-α-smooth muscle actin (03-61001) from ARP, anti-Nox4 (AF8158) from R&D systems (Minneapolies, MN), anti-Col1A1(cat# GTX82720) from Genetex (Irvine, CA); anti-survivin (#2808), anti-β-actin (#2128), and anti-H3 (#9715) were from Cell signaling (Beverly, MA), anti-H4K16Ac(#61529) and anti-H4K20Me3 (#39671) were from Active Motif (Carlsbad, CA). Immunoblots were imaged with an Amersham Biosciences 600 Imager (GE Healthcare). Densitometry analysis was done using Image J software.
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