Protein was extracted from frozen liver samples or cultured hepatocytes in cell lysis buffer. In total, 40–60 μg of protein was loaded onto a 10% SDS-polyacrylamide gel, and separated proteins were transferred to PVDF membranes. Western blot assays were performed using specific antibodies. Anti-Sirt1 antibody was purchased from Millipore (Cat. No. 7131, Millipore, Schwalbach, Germany), and anti-p-AMPKα and AMPKα antibodies were purchased from Cell Signaling Technologies (Cat. No. 2535 and 5831, Beverly, MA, USA). Anti-Srebp-1 antibody was obtained from Santa Cruz Biotechnology (Cat. No. sc-13551, Santa Cruz, CA, USA). Anti-Gapdh and α-tubulin antibodies were obtained from Abmart (Cat. No. CW0100A and CW0098A, Arlington, MA, USA). Anti-Sod2, NFκb and LKB1 antibodies were purchased from ABclonal Technology (Cat. No. A1340, A2771 and A2122, Abclonal, Wuhan, China). Anti-HDAC3 antibody was obtained from Abcam (Cat. No. ab7030, Abcam, Cambridge, UK).
Anti hdac3 antibody
Manufactured by Abcam
Sourced in United States
Anti-HDAC3 antibody is a laboratory reagent used to detect and study the HDAC3 protein in biological samples. It is a specific antibody that binds to the HDAC3 protein, allowing its identification and quantification using various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Hdac assay kit, by Active Motif (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Anti srebp 1 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti sirt 1 antibody, by Merck Group (1 mentions)
Anti phospho stat3, by Abcam (1 mentions)
Anti gpr43 antibody, by Abcam (1 mentions)
Anti claudin 1, by Thermo Fisher Scientific (1 mentions)
Anti occludin, by Thermo Fisher Scientific (1 mentions)
Anti zo 1, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid kit, by Beyotime (1 mentions)
Horseradish peroxidase conjugated goat anti mouse rabbit antibody, by CoWin Biotech (1 mentions)
Anti hdac1 antibody, by Abcam (1 mentions)
Anti hdac5 antibody, by Abcam (1 mentions)
Anti hdac 2 antibody, by Abcam (1 mentions)
Anti dnmt1 antibody, by Santa Cruz Biotechnology (1 mentions)
Confocal petri dish, by NEST Biotechnology (1 mentions)
Alexa fluor 594 r37119, by Thermo Fisher Scientific (1 mentions)
Ez chip kit, by Merck Group (1 mentions)
Anti rna polymerase 2 antibody, by Merck Group (1 mentions)
Anti pparγ antibody, by Cell Signaling Technology (1 mentions)
8 protocols using anti hdac3 antibody
1
Western Blot Analysis of Liver Proteins
2
Immunohistochemical Analysis of HDAC3 in ACP
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Paraffin-embedded tissue sections from 12 patients with pathologically confirmed ACP were obtained from the pathology department of the First Affiliated Hospital of Fujian Medical University. 5 male and 7 female patients were enrolled and the average age of the patients at the time of surgery was 46.42 ± 16.02 years old (range 14–65 years). These patients underwent surgery from July 2017 to February 2020. Eight of the patients had been confirmed to have obvious tumor calcification while the rest of four patients with no tumor calcification by computed tomography (CT) scan, as described in previous reported [15 (link)]. The paraffin sections were stained with anti-HDAC3 antibody (1:100, Abcam, USA) as previously reported [16 (link)].
3
Cecum Protein Extraction and Western Blot
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The proteins (n = 5) of cecum were extracted with RIPA lysis buffer, and the concentration with bicinchoninic acid kit (Beyotime, Wuhan, China) was determined. Then, equal amounts of protein in each group were added to SDS-polyacrylamide gel, transferred onto polyvinylidene fluoride membranes, and blocked for 1 h using 5% skimmed milk. Subsequently, primary antibodies including anti-claudin-1 (rabbit, 1:1,000, Invitrogen, California, USA), anti-occludin (rabbit, 1:1,000, Invitrogen, California, USA), anti-ZO-1 (rabbit, 1:1,000, Invitrogen, California, USA), anti-phospho-STAT3 (1:1,000, Abcam, Cambridge, UK), anti-cyclin D1 antibody (mouse, 1:200, Abbexa, Cambridge, UK), anti-HDAC3 antibody (rabbit, 1:1,000, Abcam, Cambridge, UK), anti-GPR43 antibody (rabbit, 1:1,000, Abcam, Cambridge, UK), or anti-β-actin (mouse, 1:4,000; Co Win Biotech Co., Inc, Beijing, China) were incubated with the membranes overnight at 4°C. Then, horseradish peroxidase-conjugated goat anti-mouse/rabbit antibody (1:8,000; Co Win Biotech Co., Inc. Beijing, China) was used to incubate the membranes for 2 h. The IOD of the target bands was measured by using ImageJ software (version 4.0.2; Scion Corp., Frederick, MD, USA) and normalized to the corresponding β-actin values. Each sample was tested in triplicate.
4
Immunostaining of Varicose Vein Epigenetics
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Immunostaining was performed as previously described [34 (link)]. The cross-sections of varicose vein F1 and F2, and normal veins were examined using immunostaining for detecting the expression of Class I HDACs (HDAC-1, -2, and -3), Class II HDACs (HDAC-5 and -7), and DNMTs (i.e., DNMT1 and DNMT3a). Briefly, sections of these veins were deparaffinized and blocked for 1 h with serum albumin dissolved in phosphate-buffered saline (5 mg/mL). The sections were incubated with either an anti-HDAC-1 antibody (Abcam, Waltham, MA, USA), anti-HDAC-2 antibody (Abcam, Waltham, MA, USA), anti-HDAC-3 antibody (Abcam, Waltham, MA, USA), anti-HDAC-5 antibody (Abcam, Waltham, MA, USA), anti-HDAC-7 antibody (Abcam, Waltham, MA, USA), anti-DNMT1 antibody (Santa Cruz, CA, USA), or anti-DNMT3a antibody (Santa Cruz, CA, USA) (1:50) for 1 h at 37 °C and subsequently with FITC-conjugated secondary antibody (1:1000) for 2 h at room temperature. The slides were mounted and the images were captured using a fluorescence microscope.
5
Immunofluorescence Staining of HDAC3
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Cells were cultured on a confocal petri dish (NEST Biotechnology Co. Ltd. China), fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature. The cells were stained with anti-HDAC3 antibody (1:100, Abcam, USA), which had been diluted 1:200 in 5% goat serum, overnight at 4 °C. The cells were subsequently stained with Alexa Fluor 594 (R37119) (1:200 dilution in PBS) (Invitrogen) at room temperature for 1 h, followed by incubation with DAPI (1:1000 dilution in PBS) for 5 min (min). The cells were then examined with a Lecia laser scanning microscope (FV1000, Olympus) at 100× magnification [18 (link)].
6
ChIP Assay for Transcription Regulators
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The ChIP assay was performed on 80–90% confluent cultures using an EZ-ChIP kit (Millipore, Burlington, MA, #17-371) and 1 μg of anti-RNA Polymerase II antibody (Millipore, #05-623B, clone CTD4H8, 1 μg/ml), 2 μg of anti-HDAC3 antibody (Abcam, #ab32369, clone Y415, 2 μg/ml), 1 μg of anti-PPAR-γ antibody (Cell Signaling, Danvers, MA, #2443 S, clone 81B8, 1 μg/ml), and 1 μg of rabbit IgG (Abcam, #ab171870, polyclonal, 1 μg/ml) per reaction. DNA-relative enrichment was determined by normalization to an input genomic DNA. All ChIP experiments were obtained from independent chromatin preparations, and all quantitative real-time PCR reactions were performed in duplicates for each sample on each amplicon. Primers for the ChIP-qPCR are listed in Supplementary Table 2 .
7
Measuring HDAC3 Deacetylase Activity
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HDAC3 deacetylase activity was measured with the HDAC Assay Kit (Active Motif 56200) according to manufacturer’s instructions on immunoprecipitated HDAC3 (as described above) from control, MHD3KO, Y298F, or WT-rescue BMDM with 1mg of starting material using anti-HDAC3 antibody (Abcam 7030). Substrate solution containing short peptides with acetylated lysine residues were added directly to the HDAC3-antibody-Protein A bead slurry complex. After incubation with developing solution, fluorescent signals from standards and samples were detected by a plate reader system (Biotek Synergy HT), and deacetylase activity was determined by the standard curve method.
8
Measuring HDAC3 Deacetylase Activity
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