Lung tissues were homogenized by a homogenizer and lysed using RIPA buffer containing a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The protein concentration of each group was determined using BCA kits. Protein samples (30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis and subsequently transferred onto PVDF membranes, which were blocked by 5% skim milk for 1 hour at room temperature. After rinsing three times, the blots were incubated overnight at 4°C with the following primary antibodies: anti-TLR4 (1:1,000; Cell Signaling Technology), anti-NF-κB p65 (1:1,000; Cell Signaling Technology), anti-phospho-IκBα (1:1,000; Cell Signaling Technology), anti-IκBα (1:1,000; Santa Cruz), and anti-Cit-H3 (1:1000, Abcam). The membranes were washed three times, incubated with horseradish peroxidase–conjugated secondary antibodies (1:3,000; Sigma-Aldrich), and then visualized by an enhanced chemiluminescence kit (ECL plus). We used anti-GAPDH (1:2,000; Cell Signaling Technology), anti-β-actin (1:10,000; Sigma-Aldrich) and anti-Histone-H3 (1:1500; Sigma) as internal controls. To measure the relative ratio of protein expression, band intensities were quantified by Image-Lab software (Bio-Rad).
Anti histone h3
Manufactured by Merck Group
Sourced in United States, Germany, China
Anti-histone H3 is a laboratory equipment product that recognizes and binds to the histone H3 protein, a core component of chromatin in eukaryotic cells. It is commonly used in various biochemical and molecular biology applications for the detection and analysis of histone H3.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (10 mentions)
Anti α tubulin, by Merck Group (4 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (3 mentions)
Anti actin, by Merck Group (3 mentions)
Anti flag, by Merck Group (3 mentions)
Anti h3k27me3, by Merck Group (3 mentions)
Anti brg1, by Santa Cruz Biotechnology (3 mentions)
Anti acetyl histone h3, by Merck Group (3 mentions)
Anti h3k9me1, by Merck Group (3 mentions)
Western lightning plus ecl, by PerkinElmer (3 mentions)
Anti h3k9ac, by Cell Signaling Technology (3 mentions)
Anti ezh2, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Anti c myc, by Abcam (2 mentions)
Anti gfp, by Roche (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Normal rabbit igg, by Cell Signaling Technology (2 mentions)
Anti α tubulin, by Cell Signaling Technology (2 mentions)
60 protocols using anti histone h3
1
Quantifying Lung Protein Biomarkers
2
Analyzing DNA Damage Response Proteins
3
Subcellular Fractionation and Protein Analysis
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Nuclear-cytoplasmic fractionation was conducted using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Life Technologies, 78833) according to the manufacturer's instructions. Protein concentration was determined using BCA protein assay reagent (Beyotime, P0010) with bovine serum albumin as the standard, and equal amounts of each cell lysate were separated by 12% SDS-PAGE. To assess the purity of fractionation, cytoplasmic, and nuclear fractions were confirmed by immunoblotting using anti-Actin (1:5000, Sigma, A5441) as a cytoplasmic marker, and anti-Histone H3(1:1000, Sigma, SAB4500352) as a nuclear marker, respectively.
4
Isolation of Intact Arabidopsis Nuclei
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Nuclei isolation from 5-day-old Arabidopsis seedlings was performed according to the previous protocol [50 (link)], which assured the isolation of high quality, intact nuclei. Briefly, Arabidopsis seedlings were chopped in the nuclei isolation buffer to release nucleus. The nuclei-containing solution was then filtered through nylon mesh filter, and the nuclei pellet was collected by centrifugation. The supernatant was retained as the cytosolic fraction [50 (link)]. Nuclear RNAs and cytosolic RNAs were extracted separately from the pellet and the supernatant by using RNeasy Plant Mini Kit (Qiagen). The intactness of isolated nuclei was examined under fluorescent microscopy after DAPI (4′,6-diamidino-2-phenylindole) staining. The purity of nuclei was confirmed by western blot of histone H3 and cytoplasmic marker protein PEPC (phosphoenolpyruvate carboxylase) [14 (link)]. Western blots using proteins extracted from the purified nucleus, the cytosolic supernatant fraction, and the whole cells were performed with anti-histone H3 (Sigma) and anti-PEPC (Agrisera) antibodies.
5
Generating GFP and FLAG Fusion Constructs
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The CDC2A- and CDC2B-GFP fusion constructs were generated by cloning genomic fragments containing the open reading frame (ORF) of these two genes into pFL2 by the yeast gap repair approach [67 (link), 68 ]. Similar approaches were used to generate the 3xFLAG fusion constructs for the FgCAK1 and CDC2B genes using the PFL7 vector. The resulting fusion constructs were confirmed by sequencing analysis and transformed in pairs into PH-1. The expression of both transforming vectors was analyzed by PCR and western blot analysis. Total proteins were isolated from strains expressing both transforming constructs and incubated with anti-FLAG M2 beads (Sigma-Aldrich, St. Louis, MO) as described [19 (link)]. Western blots of total proteins and proteins eluted from anti-FLAG M2 beads were detected with anti-GFP (Roche, Indianapolis, IN), anti-FLAG (Sigma-Aldrich), and anti-Histone H3 (Sigma-Aldrich) antibodies as described [72 (link)].
6
Cell Culture and Protein Analysis
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NB4, HL60, K562, Jurkat, and U937 cell lines were maintained in RPMI 1640 medium supplemented with 10% of fetal bovine serum (FBS, Gibco, Paisley, UK), 1% of L-glutamine (Lonza, Verviers, Belgium) and 1% of penicillin/streptomycin (Euroclone, Pero, MI, Italy) in a humidified atmosphere at 37 °C and 5% of CO2. Cell lines were originally obtained from ATCC repository and routinely tested by PCR method and MycoAlert (Lonza, Verviers, Belgium #LT07-318) for mycoplasma contamination by the European Institute of Oncology (Milan, Italy).
Maltonis was synthesized as already described [20 (link)] and used for the treatments of cells (stock solution of 10 mM diluted in distilled water) at the reported concentrations for 24 h [19 (link)]. Western blot analyses were performed as previously reported [23 (link)] using the following antibodies: anti-H3K9me3 (#39766, Active Motif, Carlsbad, CA, USA, 1:1000 dilution), anti-α-tubulin (#T9026, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany, 1:500 dilution), anti-Histone H3 (#05-499, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany, 1:500 dilution), anti-c-MYC (Y69, Abcam, Cambridge, MA, USA, 1:1000 dilution), and images acquired using Vü-C Imaging system (PopBio, Cambridge, UK).
Maltonis was synthesized as already described [20 (link)] and used for the treatments of cells (stock solution of 10 mM diluted in distilled water) at the reported concentrations for 24 h [19 (link)]. Western blot analyses were performed as previously reported [23 (link)] using the following antibodies: anti-H3K9me3 (#39766, Active Motif, Carlsbad, CA, USA, 1:1000 dilution), anti-α-tubulin (#T9026, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany, 1:500 dilution), anti-Histone H3 (#05-499, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany, 1:500 dilution), anti-c-MYC (Y69, Abcam, Cambridge, MA, USA, 1:1000 dilution), and images acquired using Vü-C Imaging system (PopBio, Cambridge, UK).
7
Chromatin Fractionation and Protein Analysis
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Chromatin fractionation experiments were performed as described before (Bellelli et al., 2014 (link)). In brief, cells in the mid-exponential phase of growth were collected by scraping in ice-cold 1x phosphate-buffered saline (PBS). Cells were then equally split and either directly resuspended in 1x LDS buffer or incubated for 10 min in ice-cold CSK buffer (10 mM PIPES, 100 mM NaCl, 1.5 mM MgCl2, 5 mM EDTA, 300 mM sucrose and 0.5% Triton X-100, protease inhibitors and phosphatase inhibitors). Chromatin-bound proteins were isolated by low speed centrifugation (3,000 rpm, 3 min at 4°C). Finally, samples were subjected to analysis by SDS-PAGE and western blotting with Anti-Strep-tag II (Abcam) and anti-histone H3 (Sigma) antibodies.
8
Western Blot Analysis of APP, LC3B, and p62
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Primary antibodies used in this study for Western blot analysis included a well-characterized homemade rabbit antiserum against the last 17 amino acids of APP, named APP-Cter-C17 (1/5000) [11 (link),12 (link),37 (link)], LC3B obtained from Cell Signaling (1/1000), p62 (Abcam, 1/2000, Cambridge, GB), and α-tubulin (Sigma, 1/10,000). The anti-histone H3 (1/10,000) used for normalization was obtained from Sigma (Saint-Louis, MO, USA). Secondary antibodies (peroxidase-labeled goat anti-rabbit IgG, 1/5000 or peroxidase-labeled horse anti-mouse IgG, 1/50,000) were obtained from Vector Laboratories (Eurobio Scientific, Les Ulis, France).
9
Quantifying Neuronal Protein Changes
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The mice were sacrificed under isoflurane anesthesia on day 20 after incision and/or alcohol treatment and the ipsilateral L4-L6 lumbar spinal cord tissues were harvested. Proteins from the lumbar spinal cord tissues were extracted as described previously.20 (link),24 (link) Nuclear fraction of the extracted protein was used for detecting CREB and its phosphorylation.24 (link) β-actin and histone H3 were used as loading controls for N-cadherin and CREB, respectively. Protein concentration was determined using the bicinchoninic acid method. The following affinity-purified antibodies were used: anti-N-cadherin (1:1000, Cat. # ab76057, Abcam, USA), anti-CREB (1:1000, Cat. # ab32515, Abcam, USA), anti-phospho-CREB-Ser133 (1:5000, Cat. # ab32096, Abcam, USA), anti-β-actin (1:200000, Cat. # A5316, Sigma, USA), and anti-histone H3 (1:3000, Cat. # 17168-1-AP, Proteintech, USA). The intensities of bands were quantified with densitometry using Image J software (NIH, USA). The intensity values of N-cadherin bands were normalized with β-actin and expressed as a ratio of N-cadherin/β-actin, and the intensity values of the phospho-CREB-Ser133 (p-CREB) were normalized with total CREB and expressed as a ratio of p-CREB/CREB. The specificity of anti-N-cadherin, anti-CREB anti-phospho-CREB-Ser133 antibodies has been validated previously.25 (link)–27 (link)
10
Western Blotting of Nuclear Proteins in Rat OPCs
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Western blot analysis was performed as previously described [27 (link), 28 (link)]. Total protein was extracted from cultured rat OPCs using cell lysis buffer supplemented with proteinase and phosphatase inhibitors. The nuclear proteins were extracted using a commercial kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The primary antibodies were anti-Foxg1 (1:800, Sigma-Aldrich), anti-GSK-3β (1:600, Sigma-Aldrich), anti-β-actin (1:1000, Santa Cruz), anti-β-catenin (1:500, Abcam), and anti-histone H3 (1:1000, Sigma-Aldrich). The band intensity was quantified using ImageJ software (NIH, Bethesda, MD, USA). Values were normalized to the β-actin/histone H3 level.
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