Five hundred thousand neutrophils mixed with C. albicans at a ratio of 1:2 were plated on coverslips and incubated at 37°C for 3 h. Coverslips were fixed in 10% formaldehyde for 15 min and permeabilized with 0.5% Triton X-100. After thorough wash, coverslips were blocked (10% FBS in PBS) and stained with anti-neutrophil elastase (abcam, 1:50) or anti-histone H3 antibody (Cell Signaling, 1:100) at 4°C overnight. Coverslips were stained with cell-permeable DNA dye Hoechst 33258 (1 μg/ml, Invitrogen) or cell-impermeable DNA dye SYTOX Orange (1 μM, Life technology) diluted in blocking buffer and left on ice for 15 min. Coverslips were then mounted by mounting gel and subject to fluorescence microscopic or confocal microscopic analysis.
Anti histone h3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
The Anti-histone H3 antibody is a laboratory reagent used to detect and analyze histone H3 proteins. Histones are core proteins that package and organize DNA within the eukaryotic cell nucleus. This antibody specifically recognizes the histone H3 protein, allowing researchers to study its presence, distribution, and modifications in various biological samples and applications.
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Lab products found in correlation
Sytox green, by Thermo Fisher Scientific (3 mentions)
Menadione, by Merck Group (2 mentions)
Hek293, by American Type Culture Collection (2 mentions)
Anti β tubulin ab6160, by Abcam (2 mentions)
Anti β actin gtx11003 anti gfp antibodies, by GeneTex (2 mentions)
M2 beads m8823, by Merck Group (2 mentions)
H2o2 solution, by Sangon (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti acetyl histone h3 antibody, by Merck Group (2 mentions)
Anti β actin antibody, by Cell Signaling Technology (2 mentions)
Anti β actin antibody, by Merck Group (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (2 mentions)
Normal rabbit igg, by Cell Signaling Technology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sytox orange, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Anti neutrophil elastase, by Abcam (1 mentions)
Proteinase inhibitor cocktail and phosphatase inhibitor, by Santa Cruz Biotechnology (1 mentions)
31 protocols using anti histone h3 antibody
1
Neutrophil-Candida Interaction Assay
2
Western Blot Analysis of Cellular Proteins
3
Chromatin Immunoprecipitation (ChIP) Assay
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ChIP assays were carried out using the Simple ChIP Enzymatic Chromatin IP kit (9003; Cell Signaling Technology). Anti-H3K27me3 antibody (1:50 dilution, 9733; Cell Signaling Technology), anti-EZH2 antibody (1:100 dilution, 5246; Cell Signaling Technology), anti-DNMT3A antibody (1:50 dilution, ab2850; Abcam), anti-E2F-1 antibody (1:100 dilution, 3742; Cell Signaling Technology), anti-histone H3 antibody (1:50 dilution, 4620; Cell Signaling Technology), anti-IgG antibody (1:1,000 dilution, 2729; Cell Signaling Technology) and anti-FOXM1 antibody (1:100 dilution, 20459; Cell Signaling Technology) were used according to the manufacturer's instructions. The antibodies were incubated overnight at 4°C. The DNMT3A, EZH2 and HAVCR2/LGALS9 promoters DNA were detected by RT-qPCR using the promoter DNA-specific primers listed in Table II . RT-qPCR was performed as described above. IgG was used as a negative control and histone H3 was used as a positive control.
4
Protein Extraction and Western Blotting Protocol
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Protein extracts were prepared as previously described [24 (link),25 (link)]. Briefly, A. aegypti total protein extracts were carried out by homogenizing adult female mosquitoes or Aag2 cells in TBS containing a protease inhibitor cocktail (Sigma). Proteins were recovered from the supernatant by centrifugation at 14.000xg, for 15 min. at 4°C. Protein concentration was determined by the Bradford Protein Assay (Bio-Rad). Western blots were carried out using secondary antibody (Immunopure goat anti-mouse, #31430). The primary monoclonal antibodies (ChIP grade) used were anti-H3 pan acetylated (Sigma-Aldrich #06–599), anti-H3K9ac (Cell Signaling Technology #9649) and Anti-H3K27ac (Cell Signaling Technology, #8173), according to the manufacture’s instructions. For all antibodies, a 1:1000 dilution was used. For normalization of the signals across the samples, an anti-histone H3 antibody (Cell Signaling Technology, #14269) was used.
5
ChIP Assay for AR, KDM6B, H3K27me3
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Chromatin immunoprecipitation (ChIP) assays were conducted by the Simple ChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology, 91820) according to the manufacturer’s instructions. Chromatin was immunoprecipitated using anti-AR (Cell Signaling Technology, 5153), anti-KDM6B (Abcam, ab38113), and anti-H3K27me3 (Abcam, ab6002) antibodies. An anti-histone H3 antibody (Cell Signaling Technology, 4620) and a normal rabbit IgG antibody (Cell Signaling Technology, 2729) were used as the positive control and the negative control, respectively. The ChIP-derived DNA was quantified using quantitative RT-PCR and the related primers are listed in the Supplemental Materials.
6
APE1 Arginine Methylation Detection
7
Immunoblotting Analysis of Protein Levels
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Immunoblotting was performed according to the standard protocol [22 (link)]. Anti-p65 antibody, anti-GFP antibody (04363–24; Nacalai Tesque, Kyoto, Japan), anti-GAPDH (#2118; Cell Signaling Technology), anti-Histone H3 antibody (#4499; Cell Signaling Technology) and anti-GST antibody (04435–84; Nacalai Tesque) were used as primary antibodies, and alkaline phosphatase-conjugated goat anti-mouse and rabbit IgG antibody (Sigma) were used as secondary antibodies. The levels of protein expression were determined by densitometry analysis of immunoblots using Image J software (1.49v; U.S. National Institutes of Health).
8
APE1 Arginine Methylation Detection
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HeLa cells (ATCC, CCL-2) were used for APE1 arginine methylation detection in cells. HEK-293 (ATCC, CRL-1573) cells were used to construct APE1 site-specific knock-in (KI) cell lines.
Anti-APE1 (ab194), anti-PRMT1 (ab12189), anti-GAPDH (ab9485), anti-COX IV (ab16056), anti-TOM20 (ab56783), anti-Mia40 (ab87033), anti-mono methyl arginine and dimethyl arginine (ab412), and anti-β tubulin (ab6160) antibodies were from Abcam (Cambridge, UK); the anti-Histone H3 antibody was from Cell Signaling Technology (Danvers, MA, USA; 4499); anti-β-actin (GTX11003) anti-GFP antibodies were from GeneTex (Irvine, CA, USA; 26673); and anti-asymmetric dimethyl-arginine (ASYM24, 07-414), anti-symmetric dimethyl-arginine (SYM10, 07-412), and anti-mitochondrial HSP70 antibodies were from Upstate (Lake Placid, NY, USA; H1830-91A). M2-beads (M8823) and menadione were purchased from Sigma (Cas No. 58-27-5), while the 30% H2O2 solution was purchased from Sangon (Shanghai, China; Cas No. 7722-84-1).
Anti-APE1 (ab194), anti-PRMT1 (ab12189), anti-GAPDH (ab9485), anti-COX IV (ab16056), anti-TOM20 (ab56783), anti-Mia40 (ab87033), anti-mono methyl arginine and dimethyl arginine (ab412), and anti-β tubulin (ab6160) antibodies were from Abcam (Cambridge, UK); the anti-Histone H3 antibody was from Cell Signaling Technology (Danvers, MA, USA; 4499); anti-β-actin (GTX11003) anti-GFP antibodies were from GeneTex (Irvine, CA, USA; 26673); and anti-asymmetric dimethyl-arginine (ASYM24, 07-414), anti-symmetric dimethyl-arginine (SYM10, 07-412), and anti-mitochondrial HSP70 antibodies were from Upstate (Lake Placid, NY, USA; H1830-91A). M2-beads (M8823) and menadione were purchased from Sigma (Cas No. 58-27-5), while the 30% H2O2 solution was purchased from Sangon (Shanghai, China; Cas No. 7722-84-1).
9
Western Blot Analysis of Histone Modifications
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Nuclear protein extracts from schistosomula or adult worms were prepared as previously described [31 (link)]. From each sample, 10 μg of each extract was loaded on 7–12% precast SDS-polyacrylamide gels (Bio-Rad). After transference, membranes were blocked with Tris-buffered saline (TBS) containing 0.1% Tween 20 and 2% bovine serum albumin (TBST/2% BSA) and then probed overnight with specific antibodies in TBS/2% BSA. Membranes were washed with TBST and incubated for 1 h in TBST/2% BSA with secondary antibody (Immunopure goat anti-mouse # 31430, Thermo Scientific, and peroxidase-labeled affinity anti-rabbit # 04-15-06, KPL). After washing the membranes in TBST, the bands were visualized and images were recorded with the Amersham Imaging System (GE Healthcare), and quantified with ImageJ software (NIH). Histone monoclonal antibodies used were anti-H3K4me1 (#5326, Cell Signaling), anti-H3K4me2 (#9725, Cell Signaling) and anti-H3K4me3 (#9727, Cell Signaling), following the manufacturer’s instructions. For all antibodies, a 1:1000 dilution was used. For normalization of the signals across the samples, anti-histone H3 antibody (#14269, Cell Signaling) was used.
10
Oxymatrine Modulates NLRP3 Inflammasome in INS-1 Cells
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The protein levels were analyzed by western blotting. The INS-1 cells were treated as follows: control; HG + PA; HG + PA + oxymatrine (1 μM); HG + PA + oxymatrine (10 μM). The cells were cultured in 6-well plates (4 × 105 cells/ml) for 24 h. In brief, the cell lysates were electrophoresed and transferred onto polyvinylidene fluoride membranes. Then, the membranes were incubated with the following primary antibodies (diluted in Tris-buffered saline with 0.1% Tween-20 [TBST] buffer): anti-NLRP3 antibody (ab214185, 1:1,000), anti-IL-1β antibody (ab205924, 1:1,000), anti-NF-κB p65 antibody (ab16502, 1:2,000), anti-Nrf2 antibody (ab89443, 1:500), anti-HO-1 antibody (ab13243, 1:2,000), anti-Keap1 antibody (ab119403, 1:1,000), anti-ASC antibody (ab180799, 1:2,000), and β-actin antibody (ab8226, 1:1,000) from Abcam (Cambridge, UK); anti-Gsdmd antibody (93709, 1:1,000) and anti-Histone H3 antibody (4499, 1:2,000) from Cell Signaling Technology (Boston, MA, USA); and anti-caspase-1 antibody (NBP1-45433, 1:1,000) from Novus Biologicals (Littleton, CO, USA). After washing, the membranes were incubated with the appropriate secondary antibody (diluted in TBST buffer) from Abcam (1:5,000, Cambridge, UK). Image Pro Plus 6.0 software (Media Cybernetics, Houston, TX, USA) was used to determine the intensity of the protein bands.
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