Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described (Chen et al., 2020a (link),b (link),c (link); Wu X. et al., 2020 (link); Yang et al., 2020b (link); Zhang et al., 2020 (link); Chen B. et al., 2021 (link); Dong et al., 2021 (link)). Nuclear proteins were extracted using the NE-PER Kit (Pierce) following manufacturer’s recommendation. Specific antibodies or pre-immune IgGs were added to and incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads (Santa Cruz). Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Western blot analyses were performed with anti-Clca2 (Proteintech, 19273-1, 1:500), anti-α-SMA (Sigma, A2547, 1:8000), anti-collagen type I (Proteintech, 14695-1, 1:2000), anti-Twist1 (Proteintech, 25465-1, 1:500), anti-HDAC1 (Santa Cruz, sc-7872, 1:1000), anti-HDAC2 (Santa Cruz, sc-7899, 1:1000), anti-HDAC3 (Santa Cruz, sc-11417, 1:1000), anti-HDAC8 (Santa Cruz, sc-11405, 1:1000), anti-FLAG (Sigma, F1804, 1:5000), and anti-β-actin (Sigma, A2228, 1:4000) antibodies.
Anti hdac3
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-HDAC3 is a laboratory reagent used to detect and quantify the presence of the HDAC3 protein. It functions as a specific antibody that binds to HDAC3, allowing for its identification and measurement in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti hdac1, by Santa Cruz Biotechnology (4 mentions)
Anti hdac2, by Santa Cruz Biotechnology (4 mentions)
Anti flag, by Merck Group (3 mentions)
Anti actin, by Merck Group (3 mentions)
Anti hdac6, by Santa Cruz Biotechnology (3 mentions)
Anti acetylated histone, by Abcam (3 mentions)
Anti cyclin d1, by Santa Cruz Biotechnology (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti β actin, by Abcam (2 mentions)
Sc 11417, by Santa Cruz Biotechnology (2 mentions)
Anti glucose regulated protein 78, by Santa Cruz Biotechnology (2 mentions)
Anti endoplasmic reticulum resident protein erp 44, by Cell Signaling Technology (2 mentions)
Anti histone h1, by Santa Cruz Biotechnology (2 mentions)
Non immune igg, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti collagen type 1, by Proteintech (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Anti α sma, by Merck Group (1 mentions)
Anti twist1, by Proteintech (1 mentions)
17 protocols using anti hdac3
1
Optimized Whole Cell and Nuclear Protein Extraction
2
Histological Analysis of Lung Development
3
Epigenetic Regulation in EMT Signaling
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PVDF membrane was obtained from Millipore (Bedford, MA, USA); anti-E-cadherin, anti-N-cadherin, anti-SET7/9, anti-OGG1/2, anti-HDAC3, LSD1 shRNA plasmid and JMJD2a shRNA plasmid were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-FLAG and anti-smooth muscle actin antibody and Phalloidin-TRITC were obtained from Sigma-Aldrich (St Louis, MO, USA); DCFDA and Alexa 488 secondary antibody were obtained from Molecular Probes; anti-H3K4me2, anti-H3K4me3, anti-H3K9me2, anti-H3K9me3, anti-H3, anti-LSD1, anti-JMJD2A, anti-SUV39H1, anti-NCoR1, anti-APE1, anti-β-actin, anti-β-tubulin antibodies, anti-IgG mouse and anti-IgG rabbit were obtained from Abcam (Cambridge, UK); anti-pSMAD2/3 (Ser 423/425) and anti-SMAD2/3 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); anti-8-oxo-dG antibodies were obtained from Trevigen Inc. (Gaithersburg, MD, USA); and the OxyDNA assay was obtained from Calbiochem (San Diego, CA, USA).
4
Piceatannol Inhibits Fibrosis Markers
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Piceatannol was purchased from Future Chem (Seoul, Korea). Anti-alpha smooth muscle actin (α-SMA; 1:1000, sc-130617), anti-CTGF (1:1000, sc-14939), anti-HDAC3 (1:1000, sc-11417), anti-HDAC4 (1:1000, sc-11418), anti-HDAC5 (1:1000, sc-133225), anti-TGF-β1 (1:1000, sc-146), anti-JNK (1:1000, sc-7345), anti-ERK1 (1:1000, sc-271269), and anti-GAPDH (1:1000, sc-32233) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies against collagen type I (1:1000, ab34710), HDAC2 (1:1000, ab12169), HDAC8 (1:1000, ab137474), and HDAC10 (1:1000, ab53096) were purchased from Abcam (Cambridge, MA, USA). Anti-fibronectin antibody (1:1000, MA5-11981) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-HDAC1 antibody (1:1000, 06–720) was purchased from Merck Millipore (Darmstadt, Germany). Anti-HDAC6 (1:1000, 7612), anti-Smad3 (1:1000, 9523), anti-Smad2 (1:1000, 3103), anti-Smad4 (1:1000, 9515), anti-p-Smad3 (1:1000, 9520), anti-p-JNK (1:1000, 9251), anti-p-p38 (1:1000, 4511), anti-p-ERK1/2 (1:1000, 4370), and anti-p38 (1:1000, 8690) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
5
Protein Expression Analysis by Western Blotting
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The changes in protein expression induced by the combination were evaluated using Western blotting. The cells were treated under the indicated conditions for 48 h, and whole-cell lysates were obtained using radioimmunoprecipitation assay buffer. Equal amounts of proteins were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After the membranes were blocked by 5% skimmed milk, they were incubated overnight with anti-acetylated histone (Abcam, Cambridge, UK), anti-cyclin D1, anti-glucose-regulated protein 78 (GRP78), anti-HDAC1, anti-HDAC2, anti-HDAC3, anti-HDAC6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-cleaved poly(ADP-ribose) polymerase (PARP), anti-endoplasmic oxidoreductin-1-like protein alpha (Ero1-Lα) (Cell Signaling Technology, Danvers, MA, USA), or anti-actin (Millipore, Billerica, MA, USA) primary antibodies. They were then incubated with horseradish-tagged secondary antibodies (Bio-Rad, Hercules, CA, USA). The bands were visualized by chemiluminescence with the ECL Plus system (GE Healthcare, Wauwatosa, WI, USA) according to the manufacturer’s instruction.
6
Immunoprecipitation of HDAC3 Complexes
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IP was carried out using the Millipore Catch and Release kit (Millipore, MA, USA). Following the instructions, 500 μg of nuclear lysates was incubated with 12 μg of anti-HDAC3 (ab7030), 10 μg of anti-HDAC3 (Upstate, 06-890), or 10 μg of anti-HDAC3 (SantaCruz, H-303) at 4°C for 3 h. Following elution, half of the immunoprecipitated sample was loaded onto an SDS-PAGE gel for downstream western blotting. Input samples represent 25 μg of total nuclear extracted protein.
7
Protein Expression Analysis in Cell Lysates
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After treatment under the indicated conditions, cells were washed with PBS, suspended in radioimmunoprecipitation (RIPA) buffer, incubated on ice for 15 min, and centrifuged at 20380 g for 12 min. Whole cell lysate was subjected to Western blot analysis as described previously.7 To analyze the expression of ubiquitinated proteins in the detergent‐insoluble fractions (pellets obtained after the protein extraction using RIPA buffer), the pellets were washed with PBS, lysed using Extraction buffer 4 in the WSE‐7421 EzSubcell Extract kit (ATTO, Tokyo, Japan), and then subjected to Western blot analysis. The following antibodies were used: anti‐cyclin D1, anti‐cyclin‐dependent kinase (CDK) 4, anti‐glucose‐regulated protein (GRP) 78, anti‐ubiquitin, anti‐histone deacetylase (HDAC) 1, anti‐HDAC3, and anti‐HDAC6 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐cleaved poly(ADP‐ribose) polymerase (PARP), anti‐HSP70, anti‐endoplasmic reticulum resident protein (ERP) 44, and anti‐endoplasmic oxidoreductin‐1‐like protein (Ero1‐L) from Cell Signaling Technology (Danvers, MA, USA); anti‐active caspase 3, anti‐NOXA, and anti‐acetylated histone from Abcam (Cambridge, UK); and anti‐actin from Millipore (Billerica, MA, USA).
8
Chromatin Immunoprecipitation of Epigenetic Regulators
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Chromatin immunoprecipitation was carried out on T‐ALL cells as described.40 DNA was immunoprecipitated with anti‐HDAC1 (sc‐7872X,) anti‐HDAC2 (sc‐7872X), anti‐HDAC3 (sc‐11417X), anti‐MeCP2 (sc‐137070), nonspecific IgG Abs (Santa Cruz Biotechnology, Dallas, TX, USA) or MBD3 (D1B8F) (Cell Signaling Technology, Danvers, MA, USA) and amplified by qRT‐PCR (Table S3 ) using SYBR Green PCR Master Mix on a StepOnePlus Real‐Time PCR System under standard conditions. Results were quantified by SYBR Green Real‐Time PCR analysis. The fold enrichment of immunoprecipitated samples was normalized on INPUT and expressed relative to the mock‐treated control (IgG). Results were visualized after separating PCR products on 3% agarose gel stained with GelGreen Nucleic Acid Staining.
9
Chromatin Immunoprecipitation (ChIP) Assay
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Chromatin immunoprecipitation (ChIP) assays were performed according to the manufacturer's instructions (Millipore). Each cross-linked sample was immunoprecipitated using anti-Hes1 (Abfrontier), anti-Hes6 (antiserum), anti-HDAC3 (Santa Cruz Biotechnology), or anti-histone H3 (acetyl K9, Abcam, Cambridge, MA, USA) antibodies and the DNA was purified by phenol/chloroform extraction followed by ethanol precipitation. For PCR, the following primers were used for the Hes1 N-box, 5'-TCCTTTTGATTGACGTTGTAGC-3' and 5'-GCACTATTCCAGGACCAAGG-3'.
10
Histone Deacetylation by HDAC3 Complex
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Synchronized HeLa S3 cell extracts from late G2 phase and mitosis were immunoprecipitated as described earlier using one of the following antibodies: anti-HDAC3 (Santa Cruz Biotechnology), anti-histone H1 (Santa Cruz Biotechnology), anti-phospho-histone H1 (Upstate), or non-immune IgG (Santa Cruz Biotechnology). They were then mixed with protein A/G-agarose beads (Santa Cruz Biotechnology). Bead immunocomplexes were washed twice with radioimmune precipitation assay buffer, followed by a wash with HDAC buffer (10 mm Tris-HCl (pH 8.0), 10 mm NaCl, 10% glycerol, and complete mini protease inhibitor mixture (Roche Life Science)). The washed bead immunocomplexes were incubated with hyperacetylated mononucleosomes in HDAC buffer at 37 °C for 40 min. After the incubation, SDS-PAGE loading buffer was added to the reaction mixture, and the reaction mixture was resolved by 15% SDS-PAGE. Western blotting analysis was performed using anti-acetyl-H3K9 (Upstate) and anti-acetyl-H4K5 antibodies (Santa Cruz Biotechnology). The HDAC assay reaction with the recombinant HDAC3-SMRT complex (Cell Sciences) served as a positive control, whereas the reaction with non-immune IgG (Santa Cruz Biotechnology) served as a negative control.
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