Protein samples were prepared from freshly dissected brain and used for semi-quantitative western blot analysis as previously described [33 (link)]. Primary antibodies were used in following conditions: anti-histone H3 (1:2,000; Abcam, Cambridge, MA), anti-acetyl histone H3 (1:2,000; Abcam, Cambridge, MA), anti-β-actin (1:10,000; Abcam, Cambridge, MA), anti-HDAC1 (1:2,000; Cell Signaling Technology, Danvers, MA), anti-HDAC2 (1:2,000; Cell Signaling Technology, Danvers, MA), anti-HDAC3 (1:2,000; Cell Signaling Technology, Danvers, MA), anti-iba1 (1:2,000; Wako, Osaka, Japan). Properly matched secondary antibodies were used before ECL reaction: anti-mouse IgG (1:2,000; Vector Laboratories, Burlingame, CA), anti-rabbit IgG (1:10,000; Vector Laboratories, Burlingame, CA).
Anti hdac3
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-HDAC3 is a lab reagent that targets the histone deacetylase 3 (HDAC3) protein. HDAC3 is an enzyme involved in the regulation of gene expression by modifying chromatin structure. The Anti-HDAC3 product is designed to detect and study the HDAC3 protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti hdac1, by Cell Signaling Technology (8 mentions)
Anti hdac2, by Cell Signaling Technology (6 mentions)
Anti hdac6, by Cell Signaling Technology (3 mentions)
Anti histone h3, by Abcam (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Pan anti acetylated lysine, by PTM Biolabs (2 mentions)
Pan anti crotonylated lysine, by PTM Biolabs (2 mentions)
Anti cd31, by Abcam (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Anti rabbit igg, by Vector Laboratories (1 mentions)
Anti acetyl histone h3, by Abcam (1 mentions)
Anti mouse igg, by Vector Laboratories (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti hdac1, by Santa Cruz Biotechnology (1 mentions)
Anti hdac2, by Abcam (1 mentions)
Anti gsdme, by Abcam (1 mentions)
Anti paxillin, by Abcam (1 mentions)
17 protocols using anti hdac3
1
Western Blot Analysis of Histone Modifications and HDAC Proteins
2
Antibodies and Reagents for Cell Analysis
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The following antibodies were used: anti-PAX8 (GeneTex, GTX101583), anti-PARP (#9532, Cell Signaling), anti-cleaved PARP (#5625, Cell Signaling), anti-GAPDH (#8884, Cell Signaling), anti-Paxillin (ab32084, Abcam), anti-HDAC1 (sc-81598, Santa Cruz), anti-HDAC2 (ab32117, Abcam), anti-HDAC3 (#3949, Cell Signaling), anti-H3K27Ac (ab4729, Abcam), anti-GSDME (ab215191, Abcam), anti-cyclin D1 (ab134175, Abcam). Alexa Fluor 594 phalloidin (A12381) was from ThermoFisher Scientific. Recombinant human FGF18 (#4082–25) was from Biovision. FGF18 ELISA kit (LS-F23007) was from Lifespan. CytoTox 96 Non-Radioactve Cytotoxicity Assay Kit (G1780) was from Promega. In vivo grade D-luciferin (P1042) was purchased from Promega. All inhibitors were bought from Selleck Chemicals. For in vitro assays, inhibitors were reconstituted in DMSO (Sigma-Aldrich) at a stock concentration of 10 mM.
3
Immunoblotting of HDAC3 in Breast Cancer
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Breast cancer tissues and adjacent-carcinoma tissues were homogenized using protein lysis buffer. Protein lysates were separated via SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% milk and incubated with primary antibodies against βactin (1: 5000, Cell Signaling #8457) and anti-HDAC3 (1: 5000, Cell Signaling #3949) at 4°C overnight. Membranes were washed with TBST and incubated with a horseradish peroxidase-conjugated anti-rabbit anti-mouse secondary antibody. Proteins of interest were detected with an enhanced chemiluminescent detection substrate. The process was performed according to a previously described protocol.
4
Mycobacterium smegmatis Infection of Macrophages
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Mycobacterium smegmatis mc2155 was grown in Middlebrook’s 7H9 broth medium (Difco, New Jersey, USA) containing 0.05% Tween 80, 0.5% glucose and 0.5% albumin at 37°C on a shaker at 120 rpm. Murine RAW264.7 macrophage cell line was cultured in Dulbecco’s Modified Eagle’s medium (DMEM; HiMedia, Mumbai, India) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 1% L-glutamine. The cells were seeded onto 24-well and 6-well culture dishes at a density of 2x105 cells/ml and 1x107 cells/ml, respectively and proceeded for experiments. Anti- ATG5, anti-ATG7, anti-Beclin1, anti- H3K9me3, anti- H3K27me3, anti- H3K9ac, anti- H3K27ac, anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-phospho–p38, anti-p62/SQSTM1, anti-GAPDH, anti-β-actin, and secondary goat anti-rabbit and goat anti-mouse antibodies were purchased from Cell Signaling Technologies (Massachusetts, USA). Anti-LC3I/II antibody was purchased from Sigma (Missouri, USA). All the pharmacological inhibitors were purchased from Sigma (Missouri, USA) and Calbiochem (Massachusetts, USA) and reconstituted in DMSO (Himedia, Mumbai, India) or sterile H2O at the following concentrations: U0126 (10 µM), SB203580 (10 µM), UNC0638 hydrate (5µM), rapamycin (50nM) and 3MA (10mM).
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Mycobacterium smegmatis mc2155 was grown in Middlebrook’s 7H9 broth medium (Difco, New Jersey, USA) containing 0.05% Tween 80, 0.5% glucose and 0.5% albumin at 37°C on a shaker at 120 rpm. Murine RAW264.7 macrophage cell line was cultured in Dulbecco’s Modified Eagle’s medium (DMEM; HiMedia, Mumbai, India) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin solution, and 1% L-glutamine. The cells were seeded onto 24-well and 6-well culture dishes at a density of 2x105 cells/ml and 1x107 cells/ml, respectively and proceeded for experiments. Anti- ATG5, anti-ATG7, anti-Beclin1, anti- H3K9me3, anti- H3K27me3, anti- H3K9ac, anti- H3K27ac, anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-phospho–p38, anti-p62/SQSTM1, anti-GAPDH, anti-β-actin, and secondary goat anti-rabbit and goat anti-mouse antibodies were purchased from Cell Signaling Technologies (Massachusetts, USA). Anti-LC3I/II antibody was purchased from Sigma (Missouri, USA). All the pharmacological inhibitors were purchased from Sigma (Missouri, USA) and Calbiochem (Massachusetts, USA) and reconstituted in DMSO (Himedia, Mumbai, India) or sterile H2O at the following concentrations: U0126 (10 µM), SB203580 (10 µM), UNC0638 hydrate (5µM), rapamycin (50nM) and 3MA (10mM).
5
Regulation of IRF7 Signaling Pathway
6
Forkhead Binding in Arg1 Promoter
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Nuclear protein extracts were prepared from RAW 267.4 cells before IL-4 treatment. A double-stranded oligonucleotide containing the consensus forkhead binding region in the Arg1 promoter area was labeled using the Biotin 3′ End DNA Labeling Kit (PIERCE). This biotin-labeled oligonucleotide probe (20 fmol) was incubated with the nuclear extracts in the presence of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 4% glycerol and 0.5 mM DTT for 20 min at room temperature using the LightShift® Chemiluminescent EMSA Kit (Thermo scientific). For the competition experiments, the nuclear extracts were incubated with 0, 1, 2 or 5 μl of anti-FoxO1 (2880, Cell Signaling), anti-HDAC3 ((3949, Cell Signaling) or anti-NCoR1 (5948, Cell Signaling)) antibody, or an equal amount of normal rabbit or IgG (sc-2027, Santa Cruz) for 60 min at room temperature. The non-denaturing binding reaction mixture was applied for electrophoresis on 6% polyacrylamide gels. The nylon membrane transferred from the gels was crosslinked using a commercial UV-light crosslinking instrument equipped with 254 nm bulbs and visualized by chemiluminescence.
7
Protein Isolation and Western Blot Analysis
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Protein isolation was performed with RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycolate, 1 mM EDTA pH 8 and 1 tablet of protease inhibitor cocktail Roche Complete (Roche) and 1 tablet of phosphatase inhibitor PhosSTOP (Roche) added per 10 ml of buffer. In total, 40 μg of protein was resolved under denaturing conditions (SDS-PAGE) and transferred to the PVDF membrane, and the membrane was blocked in Odyssey blocking buffer (Li-Cor Bioscience). Blots were probed with anti-HDAC3 (cat no 2632, Cell Signalling Denver, CO, 1:1000 or Abcam, Cambridge UK, AB93172, 1:1000), anti-HSD17B4 (Novus Biologicals, Colorado, US, NPB1-33192, 1:2500), anti-SCD1 (Cell Signaling, CST2784s, 1:2500) at 4 °C overnight or anti-β-actin (cat no A5316, Sigma-Aldrich, St. Louis, MO, 1: 10,000) at room temperature 1 h. Secondary antibodies used were IRDye® 800CW Goat anti-Mouse IgG (P/N: 926-32210, 1:10,000) and IRDye® 680RD goat anti-rabbit IgG (P/N: 926-68071, 1:10,000, Li-Cor Bioscience, Nebraska, USA). The infrared fluorescence image was obtained using Odyssey infrared imaging system (Li-Cor Bioscience). Uncropped blots are available in the source data file.
8
Western Blot Analysis of Histone Deacetylases
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Equivalent amounts of protein (10–20 µg) from retina lysates were separated in a Nu PAGE 10% Bis-Tris gel (Thermo Fisher Scientific, Carlsbad, CA, USA) and proteins were transferred to nitrocellulose membranes, as described earlier.8 (link),22 (link) The membranes were blocked with 5% non-fat dry milk for 1 hour followed by incubation with primary antibodies (anti-HDAC 1 [Cell Signaling Technology; Cat # D5C6U], anti-HDAC 2 [Cell Signaling Technology; Cat # D6S5P], anti-HDAC 3 [Cell Signaling Technology; Cat # 7G6C5], or anti-HDAC 6 [Cell Signaling Technology; Cat # D21B10] at 1:1000 dilutions, or β-actin [Sigma Aldrich; Cat # A5316] at 1:4000 dilutions) for 16 hours at 4°C. After washing, the membranes were incubated for 1 hour at room temperature with the appropriate secondary antibodies (Anti-rabbit IgG, HRP-linked; Cell Signaling Technology; Cat # 7074; anti-mouse IgG, HRP-linked; Cell Signaling Technology; Cat # 7076 dilution 1:5000). Prestained molecular weight magic markers (Thermo Fisher Scientific) were run along with the samples to identify the molecular weight of proteins. For chemiluminescent detection, the membranes were treated with enhanced chemiluminescent (ECL) reagent for 1 minute (Super Signal; Thermo Scientific, Rockford, IL, USA), and the signal was captured using a Biorad Versadoc imaging system (Biorad, Hercules, CA, USA).
9
Western Blot Analysis of Circadian Proteins
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Western blot analysis was performed as previously described with some modifications [18] (link). Cells were lysed in whole-cell lysis buffer A [50 mM Tris–HCl (pH 7.4), 1 mM DTT, 1% (v/v) Triton X-100, and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan)]. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) was used to separate 20 μg of lysates, which were then transferred to polyvinylidene difluoride membranes. The following primary antibodies were used to probe the blots: anti-ALAS1 (Abcam, Cambridge, UK, ab54758), anti-BMAL1 (Abcam, Cambridge, UK, ab3350, [19] (link)), anti-HO-1 (Abcam, Cambridge, UK, ab13248, [20] (link)), anti-NR1D1 (Abcam, Cambridge, UK, ab56754, [21] (link)), anti-ACTIN (MP Biomedicals, Irvine, CA, USA, 69100), and anti-HDAC3 (Cell Signaling Technology, Beverly, MA, USA, 3949). For the second antibody, we used anti-mouse IgG HRP-conjugated antibody (Cell Signaling Technology, Beverly, MA, USA) and anti-rabbit IgG HRP conjugates (Santa Cruz Biotechnology) at 1:3000 dilution.
10
Western Blot Antibody Validation
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Western blot was performed as described previously [47 (link)]. The following antibodies were used. Anti-GAPDH and Anti-YWHAZ were purchased from Proteintech Company (Chicago, IL, USA). Anti-HDAC3, Anti-HDAC1, Anti-p-AKT, Anti-AKT, Anti-c-Myc and Anti-IgG were purchased from Cell Signaling Technology.
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