Total protein content was determined by BCA assay (Beyotime, Beijing, China). Then, the protein specimens were separated through 10% SDS-PAGE and transferred onto a PVDF membrane. After blocking with skimmed milk, the membrane was incubated with appropriate antibodies overnight at 4°C. The following primary antibodies were obtained from Abcam: HDAC1 (1:1000; ab109411), HDAC2 (1:1000; ab32117), HDAC3 (1:1000; ab32369), HDAC6 (1:1000; ab1440), AKT (1:1000; ab8805), p-AKT (1:500; ab38449), eNOS (1:1000; ab199956), p-eNOS (1:500; ab215717), FoxO3a (1:1000; ab70315), p-FoxO3a (1:500; ab47285), CD31 (1:1000; ab281583), α-SMA (1:1000; ab5694), and FGF21 (1:1000; ab171941). The primary antibody GAPDH (1:3000; 10494-1-AP) was purchased from Proteintech Group (Chicago, USA). After incubation with the primary antibodies, the membrane was rinsed 3 times and incubated with horseradish peroxidase–conjugated secondary antibody goat anti-rabbit immunoglobulin (1:5000; Zhongshanjinqiao, China). Finally, the membranes were visualized using an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology), and their intensities were determined with ImageJ software.
Hdac1
Manufactured by Abcam
Sourced in United States, United Kingdom, China
HDAC1 is a histone deacetylase enzyme that removes acetyl groups from histone proteins, which can lead to transcriptional repression. It is involved in the regulation of gene expression and plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Hdac2, by Abcam (16 mentions)
Hdac3, by Abcam (7 mentions)
β actin, by Merck Group (5 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Ecl plus reagent, by Thermo Fisher Scientific (3 mentions)
Dnmt3a, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Gapdh, by Bioworld Technology (2 mentions)
Anti atp5a, by Abcam (2 mentions)
Anti darpp32, by Abcam (2 mentions)
Anti htt epr5526, by Abcam (2 mentions)
Anti polyq php3, by Abcam (2 mentions)
Anti pde10a, by Abcam (2 mentions)
Anti vinculin, by Merck Group (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti gfap, by Merck Group (2 mentions)
Anti polyq mw1, by Merck Group (2 mentions)
Anti htt mw8, by Developmental Studies Hybridoma Bank (2 mentions)
Dnmt1, by Abcam (2 mentions)
54 protocols using hdac1
1
Quantitative Protein Expression Analysis
2
Investigating HDAC1, HSP90, NFKB2, IKKB, and TNF-α Expression
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Western blotting and semi-quantitative analysis were performed as in our previous study [36 (link)]. To investigate the effect of GSZD on the expression levels of HDAC1, HSP90AA1, NFKB2, IKBKB and TNF-α proteins, HFLS were treated with 10 ng/mL of IL-1β in the presence of various concentrations of GSZD. Antibodies against the following proteins were used: HDAC1 (rabbit polyclonal antibody; dilution 1:1000; Abcam, Cambridge, UK), HSP90AA1 (rabbit polyclonal antibody; dilution 1:500; Abcam, Cambridge, UK), NFKB2 (rabbit monoclonal antibody; dilution 1:1000; Cell Signaling), IKKB (rabbit monoclonal antibody; dilution 1:1000; Abcam, Cambridge, UK), and TNF-α (rabbit polyclonal antibody; dilution 1:100; Abcam, Cambridge, UK). All experiments were performed in triplicate. The mean normalized protein expression ± SD was calculated from independent experiments.
3
Histone Acetylation and AhR Binding Analysis
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ChIP assay was conducted to determine the acetylation status of histone 3 in the promoter region of the SHC1 gene and the enrichment of Ahr in the promoter region of the LOC101928120 gene. A Magna ChIP Kit (Millipore, Bedford, MA, USA) was used to extract nuclear DNA-protein complex in chondrocytes. The nuclear DNA was sonicated to produce 200–500 bp fragments. The chromatin extract was incubated with antibodies against HDAC1 (Abcam), acetylated histone 3 (Abcam), Ahr (Abcam), or IgG (Millipore). After immunoprecipitation, the DNA-protein-antibody complex was separated and the protein was removed. The purified DNA was then analyzed using qRT-PCR. The IgG and input groups were used as the negative and positive control groups, respectively.
4
Western Blot Analysis of Apoptosis Markers
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Cells were lysed with 0.5% NP40 lysis buffer and western blotting was performed according to the standard protocol. Detection was accomplished with the chemiluminescence ECL plus reagent (Thermo, Grand Island, NY, USA) and the chemiluminescence HRP substrate (Millipore, Billerica, MA, USA), and signals were evaluated by a Tanon 5200Multi scanner (Shanghai, China). Primary antibodies were as follows: HDAC1 (Abcam, Cambridge, UK), HDAC2 (Abcam), HDAC3 (Abcam), cleaved caspase-3 (CST, Danvers, MA, USA), cleaved PARP (CST), PARP (CST), GAPDH (Bioworld, St. Louis Park, MN, USA), K9 acetyl-histone H3 (CST), Bax (CST), P53 (Santa Cruz), PUMA (CST), and HA (CST).
5
Antibodies and Dilutions for Huntington's Disease
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The following antibodies and dilutions were used in this study: Anti-HTT Ab1 (aa1–17,1 (link)) 1:50 for capillary immunoassay and 1:2000 for western blot; anti-HTT EPR5526 (Abcam, Waltham, MA, ab109115, 1:2000 for western blot); anti-polyQ MW1 (MilliporeSigma, Burlington, MA, MABN2427, 1:50 for capillary immunoassay); anti-polyQ PHP3 (generous gift from Dr. Ali Khoshnan, 1:2000 for western blot); Anti-PDE10A (Abcam, Waltham, MA, #ab177933, 1:2000 for western blot); Anti-DARPP32 (Abcam, #ab40801, 1:2000 for capillary immunoassay); Anti-GFAP (MilliporeSigma, Burlington, MA, AB5804, 1:3000 for capillary immunoassay); Anti-GAPDH (MilliporeSigma, Burlington, MA, #MAB374, 1:10000 for western blot); Anti-Sodium channel subunit beta-4 (Abcam, Waltham, MA, #ab80539, 1:500 for western blot); Anti-vinculin (Sigma, St. Louis, MO, #V9131, 1:5000 for capillary immunoassay, 1:2000 for western blot); Anti-ATP5A (Abcam, Waltham, MA, #ab14748, 1:2000 for western blot); Anti-HTT MW8 (University of Iowa Developmental Studies Hybridoma Bank, 1:1000 for filter trap); Anti-HTT S830 (generous gift from Dr. Gillian Bates, 61 (link) 1:8000), HDAC1 (Abcam, Waltham, MA, ab32369–7, 1:4000).
6
Protein Expression Analysis of Cardiac Remodeling
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Scar and LV tissue were dissected from AMI, CHF, and sham hearts, respectively. Dissected tissues and isolated cells were homogenized in lysis buffer (50 mM Tris- HCl pH7.5, 150 mM NaCl, 0.5% NP-40 (Sigma), 0.5% Triton-X (Sigma), 1 mM EDTA (Sigma), and complete mini protease inhibitor (Roche). Protein concentrations were determined by BCA assay (Thermo Scientific). Typically, 40 μg of protein was loaded on 4% to 12% Tris-Bis gels (Life Technologies), separated in MOPS running buffer, and transferred to a PVDF membrane (Millipore). After blocking with 5% non-fat dry milk in 1× TBS, membranes were probed with HDAC1, HDAC2 (Abcam, 1:1,000), α-SMA (Sigma, 1:5,000), E-cadherin, GSK3β (Santa Cruz, 1:500), p-GSK3β, Cleaved Caspase 3, p-Akt, Akt (Cell Signaling, 1:1,000), β-Catenin active (Millipore,1:1,000), and β-Actin (Sigma, 1:50,000) in TBS with 5% milk overnight at 4°C. Following three washes in TBS, membranes were incubated with HRP-conjugated secondary antibodies (Santa Cruz, 1:50,000) for 1 h at room temperature. An ECL (Millipore) system was used for detection of the bands and exposed to X-ray film (Thermo Scientific) in a dark room. Densitometry analysis was performed with Alpha Ease FC software.
7
Myocardial Protein Quantification and Analysis
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The total protein in myocardial tissues was extracted, and the protein concentration was determined by the bicinchoninic acid method. The protein was transferred to the polyvinylidene fluoride membrane and added with blocking fluid (PBS containing 3% fetal bovine serum and 0.1% Tween-20) for 1 h. The membrane was probed with the corresponding dilution proportion (all 1:1000) of primary antibodies HDAC1, HMGB1 (Abcam), β-actin (Santa Cruz Biotechnology, CA, USA) and cleaved caspase-3 (Abcam) overnight, then re-probed with immunoglobulin G (Cell Signaling Technology) labeled by horseradish peroxidase. The blots were developed using enhanced chemiluminescence (ECL) Kit from Beyotime Institute of Biotechnology (China) and visualized using the ChemImager 5500 V2.03 software from Alpha Innotech (San Leandro, CA).
8
CUT&RUN Profiling of Histone Deacetylases in Forelimb Development
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Experiments were performed according to EpiCypher’s CUTANA CUT&RUN protocol (EpiCypher #15-1016) with the following modifications. 200,000–300,000 cells from E9.25 (21–23 S) forelimbs and anterior E10.5 (32–35 S) forelimbs were incubated overnight at 4 °C on a nutator with 1:250 FLAG primary antibody (Sigma #F3165), 1:100 HDAC1 (Abcam ab7028) or 1:200 HDAC2 (Abcam ab7029). The following day, samples were incubated at room temperature for 30 min. with secondary antibody, 1:100 Donkey anti-mouse (Jackson ImmunoResearch #715-035-150) or Donkey anti-rabbit (Jackson ImmunoResearch #711-005-152), followed by three washes in Digitonin wash buffer. CUTANA pAG-MNase was then incubated with samples for 10 min at room temperature and then the MNase reaction was performed for 2 h at 4 °C on a nutator. Libraries were generated using NEBNext Ultra II DNA Library Prep Kit with 14 PCR cycles and cleaned up to remove adapters using AMPure XP beads (Beckman Coulter) or SparQ PureMag beads (QuantaBio). Samples were sequenced on an Illumina NextSeq 500 instrument using PEx75 to a depth of depth of 3–5 million reads. Peaks were called using MACS79 (link).
9
ChIP-seq analysis of AML1, ETO, and epigenetic regulators
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Formaldehyde (1% final concentration) was added into cells (2 × 106 cells). Cells were then incubated for 10 min at 37°C to crosslink proteins to DNA. After sonication, 5 μg of antibodies recognizing the following AML1 (Santa Cruz Biotechnology, Santa Cruz, USA, sc-28679), ETO (Santa Cruz Biotechnology, Santa Cruz, USA, sc-9737), histone deacetylase 1 (HDAC1) (Abcam, Cambridge, USA, ab7028), DNA methyltransferase 1 (DNMT1) (Abcam, Cambridge, USA, ab13537), DNMT3a (Abcam, Cambridge, USA, ab13888) and DNMT3b (Abcam, Cambridge, USA, ab13604) were immunoprecipitated with the chromatin overnight. Chromatin immunoprecipitation (ChIP) was performed on the naked and sonicated DNA extracted from SKNO-1, SKNO-1-siA/E, U937, and U937-A/E cell lines and then assayed with the EZ-ChIP kit (Millipore, Billerica, MA, USA, 17-371) according to the instructions of the manufacturer. Genomic EYA4 upstream regions, which were close to the putative AML1-binding site, were amplified. Primers sequences are shown in Supplementary Table 1 . GAPDH was used as a control for nonspecific precipitated sequences.
10
Immunohistochemical Analysis of HDAC1 Expression
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The expression of HDAC1 protein within tissues were verified by IHC as previously described (6 (link)). A total of 8 cases of tissue (4 cases in medulloblastoma and 4 cases in AT/RT) used to verify HDAC1 protein expression. Of these, 3 cases of each group were newly obtained, and 1 case of each group was included in the previous RT-qPCR analysis. Briefly, the sections were incubated with primary antibodies, HDAC1 (1:1000, Abcam, Cambridge, MA), for 32 min at 37°C, and a secondary antibody for 20 min at 37°C. The stained sections were detected using the Ventana ChromoMap Kit (Ventana Medical Systems) and discovered using XT automated IHC strainer (Ventana Medical Systems, Oro Valley, AZ).
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