Protein lysates were prepared as previously described [11 ]. Briefly, 50 mg of the liver was homogenized in 1 mL of cell lysis buffer, 1X proteinase inhibitor, and 1X PMSF. Samples were sonicated on power 4, thrice for 5 s each, and then, spun at 13,200 RPMs for 15 min at 4 °C. Protein concentrations were determined using a BCA reagent (23,227; Pierce™). Protein lysates were resolved in 3–8% Tris-Acetate Novex gel (Invitrogen) for NCOR1, SMRT/NCOR2, and beta-actin or 10% Bis-Tris gel (Invitrogen) for hdac3 and hsp90, and then, transferred to a nitrocellulose membrane. Blots were probed for NCOR1 using rabbit polyclonal anti-NCOR1 antibody (PHQQ; generated against a C-terminal NCoR peptide as previously described; diluted at 1:250), rabbit polyclonal anti-SMRTe (06–891; Millipore Sigma; diluted at 1:500), or rabbit polyclonal anti-HDAC3 (ab7030; abcam; diluted at 1:2000) overnight at 4 °C with mouse monoclonal anti-beta-actin antibody (MA191399; Fisher Scientific; diluted at 1:10,000) for NCOR1 and SMRT/NCOR2 control or mouse monoclonal anti-hsp90a/b antibody (sc-13119, Santa Cruz Biotechnology; 1:5,000). Goat anti-rabbit Alexa Fluor Plus 680 (A32734; Invitrogen) and goat anti-mouse Alexa Fluor Plus 488 (A32723; Invitrogen) were used as secondary antibodies and incubated at room temperature for 90 min. Images were captured on ChemiDoc Touch MP (Bio-rad).
Ab7030
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab7030 is a lab equipment product offered by Abcam. It is designed to perform a specific function in a laboratory setting. The core function of this product is to [DESCRIPTION_NOT_AVAILABLE].
Automatically generated - may contain errors
Lab products found in correlation
Ab12723, by Abcam (2 mentions)
Ab7028, by Abcam (2 mentions)
Ab12169, by Abcam (2 mentions)
Ab109489, by Abcam (2 mentions)
Wrab 1200, by Abcam (2 mentions)
Ab3482, by Abcam (2 mentions)
Ab180904, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
Ecl western blotting detection reagent, by GE Healthcare (2 mentions)
Alexa fluor plus 488, by Thermo Fisher Scientific (1 mentions)
Sc 13119, by Santa Cruz Biotechnology (1 mentions)
Chemidoc touch mp, by Bio-Rad (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab93806, by Abcam (1 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (1 mentions)
Nxtract, by Merck Group (1 mentions)
Ab96777, by Abcam (1 mentions)
Ab28146, by Abcam (1 mentions)
Ab62352, by Abcam (1 mentions)
34 protocols using ab7030
1
Protein Lysate Preparation and Western Blotting
2
Immunofluorescence Visualization of HDAC3
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MCF7 cell were cultured in complete medium for 48 hours and fixed in 4% paraformaldehyde for 10 minutes at room temperature. The cells were permeabilized with phosphate-buffered saline (PBS) containing 0.2% Triton-X100 for 5 minutes at room temperature, washed three times in PBS containing Triton-X100 and blocked with 5% bovine serum albumin for 30 minutes at room temperature. The cells were incubated with antibody against HDAC3 (ab7030; Abcam) diluted 1:200, overnight at 4℃. After washing, the cells were labeled with secondary antibody, followed by examination under a fluorescence microscope (Nikon Corp., Tokyo, Japan). The nuclear dye 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Sigma-Aldrich (Darmstadt, Germany) and was incubated with the immunostained cells at room temperature for 10 minutes at the concentration of 1 µg/mL.
3
Muscle Transcription Factor Regulation Profiling
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In vivo skeletal muscle ChIP was performed with sonicated nuclear extract prepared from formaldehyde-cross-linked gastrocnemius muscle according to [137 (link)]. For immunoprecipitation, we used anti-BMAL1 (ab93806, Abcam), anti-REV-ERBα (generous gift from Ron Evans), anti-RNA Polymerase II (#MMS-126R; clone 8WG1, Biolegends), anti-NCOR1 (#20018-1-AP, Protein Tech), anti-HDAC3 (ab7030, Abcam), or rabbit IgG (2027x, Santa Cruz). DNA was column purified and used for sequencing or real-time qPCR (enrichment expressed as fold-change relative to IgG; primer sequences used are listed in S4 Table ).
4
HDAC3 Immunohistochemistry in Paraffin Sections
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The paraffin-embedded sections were deparaffinized with xylene, followed by rehydration using a graded series of ethanol, and then stained with anti-HDAC3 antibody (ab7030; Abcam, Cambridge, USA) diluted 1:500. Sections were subsequently incubated for 1 hour with biotinylated IgG as a secondary antibody and then for 30 minutes with horseradish peroxidase (HRP)-conjugated streptavidin. Colorimetric reaction products were visualized using 3,3′-diaminobenzidine as a chromogen. All sections were counterstained with hematoxylin.
5
Nuclear Extract Binding Assay Protocol
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Nuclear extracts were prepared from EL4 cells using NXTRACT (Sigma-Aldrich). In binding assays of 20 μl total volume, 28 μg of total nuclear extract from EL4 cells was incubated with 20 fmol of 3′ biotin-labeled oligonucleotide in the presence of 2 μl of 10× binding buffer (100 mM Tris, 500 mM KCl, 10 mM DTT [pH 7.5]) and 10 μM ZnSO4, 1 μl of 50% glycerol, 1 μg/μl poly(deoxyinosinic-deoxycytidylic) acid, 5 mmol/L MgCl2 and 1% Nonidet P-40 at room temperature for 20 minutes. The oligonucleotides and methylated oligonucleotides (eurofins) were annealed and used as probes or competitors (Supplementary Table 2 ). All competitors were used at 200-fold excess. For supershift experiments, the nuclear extracts were incubated with 4 μg of anti-Kaiso Ab (6 F/6F8, ab12723; Abcam) and 5 μg of anti- HDAC3 Ab (ab7030; Abcam). The binding mixtures were loaded onto 6% native acrylamide gel (Thermo Fisher Scientific) in Tris/borate/EDTA buffer and electrophoresed for 50 minutes at room temperature under a constant 100 V. The gels were then transferred to a nylon membrane at 4 °C for 50 minutes under a constant 100V and exposed to UV light to crosslink for 15 minutes. The DNA binding activity was detected using a LightShift chemiluminescent EMSA kit (Thermo Fisher Scientific). The image was obtained using an LAS-3000 IR.
6
Romidepsin and Lipopolysaccharide Interactions
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Romidepsin (FK228) (S3020), was purchased from Shanghai Selleck Chemicals Co., Ltd. (Shanghai, China). Lipopolysaccharide (LPS) derived from Escherichia coli 055: B5 (L2637) was purchased from Sigma-Aldrich (St Louis, MO, USA). Antibodies to acetyl-histone H3 (4499) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-KIM-1 (AF1817) was purchased from R&D Systems (Chengdu, China). Antibodies to CYP2E1 (ab28146), HDAC1 (ab7028), HDAC2 (ab12169), HDAC3 (ab7030), hepatocyte nuclear factor-1 alpha (HNF-1α) (ab96777) and Nrf2 (ab62352) were purchased from Abcam (Cambridge, UK). Anti-GAPDH (TA-08) was purchased from ZSGB Biotechnology (Beijing, China).
7
ChIP-seq analysis of HDAC3 in miR-494 promoter
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Fresh ESCC and adjacent tissues were cut into 1–3 mm3 small pieces and transferred to a 50 mL test tube. Next, the tissues were fixed with formaldehyde at ambient temperature for 10 min for generating DNA–protein crosslink. The reaction was terminated by 2.5 M Glycine (final concentration of 0.125 M). The samples were lysed using a homogenizer, sub-packed (1 mL per tube), re-suspended, and fragmented by ultrasonic wave, 10 s each at an interval of 10 s, 15 cycles in total. The supernatant was harvested through centrifugation at 13,000 rpm and 4ºC for 10 min and sub-packed into 3 tubes. The tubes were respectively added with normal mouse antibody to IgG (ab109489, 1: 100, Abcam) as NC and mouse antibody to HDAC3 (ab7030, 1: 100, Abcam) for incubation at 4ºC overnight. The endogenous DNA–protein complex was precipitated by Protein Agarose/Sepharose. The non-specific complex was washed and the DNA–protein complex was incubated at 65ºC overnight for de-crosslinking. DNA fragment was purified and retrieved by phenol/chloroform. The primer was designed by selecting 2000 bp from the transcription start site to the upstream as the promoter sequence. The enrichment of HDAC3 in the miR-494 promoter region was tested by RT-qPCR [29 (link)].
8
Chromatin Profiling of Foxa2 and Hdac3
9
Western Blot Analysis of Testicular Proteins
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Western blot experiments on whole testes were performed as described in Comptour et al. (2014) (link) and Moretti et al. (2017) (link). Fifteen microliters of immunoprecipitated samples and 5 µl of input were denatured in 1× NuPAGE LDS sample buffer (Life Technologies) at 95 °C during 10 min. Antibody against SLY (Reynard et al. 2009 (link)) and against SLX/SLXL1 (Reynard et al. 2007 (link)) was diluted 1/3,000; anti-TUBULIN was diluted 1/5,000 (05-661, Millipore); and anti-TBLX1R1 (ab24550, Abcam), anti-HDAC3 (ab7030, Abcam), anti-TBL1X (ab24548, Abcam), anti-SSTY1 (Comptour et al. 2014 (link)), anti-SPIN1 (orb193004, Biorbyt), and anti-NCor1 (ab3482, Abcam) were diluted 1/1,000.
10
Immunohistochemical Analysis of HDAC3 and IL17RA in Lung Tissues
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Human lung tissues from controls, patients with IPF, and patients with RA-ILD were fixed with 4% paraformaldehyde, mounted to conventional paraffin embedding, sliced serially as 4 μM sections, and dewaxed. After antigen retrieval, normal goat serum was added for blocking. The lung tissues of control patients were used as negative controls during the analysis, and stained with Histostain™ SP-9000 immunohistochemical staining kit (Zymed Laboratories, San Francisco, CA, United States). The tissues were added with primary antibody rabbit anti HDAC3 (ab7030, 1:500, Abcam) and rabbit anti IL17RA (ab180904, 1:500, Abcam). After incubating with secondary antibody (ab9482, 1:5,000, Abcam) at 37°C for 30 min, horseradish-labeled working fluid was added to the tissues, and the tissues were developed by diaminobenzidine. The tissues were counterstained with hematoxylin for 1 min, and photos were taken after mounting. Five representative high power fields (positive optical microscope, NIKON, Tokyo, Japan) were selected for counting the cells with brown or yellow cytoplasm to calculate the positive rate.
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