Before the collection of protein extracts, cells were dissolved in lysis buffer. The cell lysis buffer was mixed with Flag M2-affinity gel-conjugated GATA1 antibody at 4°C for 12 h. After that, the protein was gradually eluted by KCl-contained buffer. The lysates (approximately 300 µg protein) were incubated with fresh protein A-beads (35 µL, #9863, CST) and 1 µg anti-GATA1 (1:1,000, ab181544, Abcam), anti-Flag (1:500, AF519, Beyotime) or IgG (1:500, ab172730, Abcam) at 4°C for 3 h. The magnetic beads were lysed in lysis buffer and loaded in SDS-PAGE. The immunoprecipitated proteins were reacted with anti-HDAC1 (1:1,000, ab109411, Abcam), anti-HDAC2 (1:1,200, ab32117, Abcam) and anti-HDAC3 (1:2,000, ab32369, Abcam). The immunoblot reactions were performed as described previously [25 (link)].
Ab32369
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab32369 is a primary antibody that recognizes human CD3 epsilon. It is a mouse monoclonal IgG1 antibody. The core function of this product is to bind to the CD3 epsilon subunit of the T cell receptor complex.
Automatically generated - may contain errors
Lab products found in correlation
Ab32117, by Abcam (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ab109411, by Abcam (3 mentions)
Ab19845, by Abcam (3 mentions)
Ab172730, by Abcam (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ab75430, by Abcam (2 mentions)
Ab6721, by Abcam (2 mentions)
Af2305, by Affinity Biosciences (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab181544, by Abcam (1 mentions)
Af519, by Beyotime (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Fv3000, by Olympus (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ab97069, by Abcam (1 mentions)
A2228, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Pa5 49431, by Thermo Fisher Scientific (1 mentions)
Ab18521, by Abcam (1 mentions)
19 protocols using ab32369
1
Immunoprecipitation and Immunoblotting for GATA1
2
Quantitative Immunofluorescence Analysis of HDAC Proteins
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Animals were intracardially perfused with PBS and 4% paraformaldehyde. After fixation, dissected brains were cryoprotected in 20% sucrose overnight at 4°C and then in 30% sucrose at 4°C. Coronal sections of 20 μm were obtained using a cryostat and further processed for immunohistochemistry. Briefly, slices were incubated in blocking fluid (Beyotime) to permeabilize and block unspecific staining. Primary antibodies were incubated for 48 h at 22–24°C and secondary antibodies overnight at 4°C. Both primary and secondary antibodies were diluted in blocking fluid (Beyotime). Rabbit anti-HDAC1 (ab19845, Abcam, 1:1,000), anti-HDAC2 (ab32117, Abcam, 1:250), and anti-HDAC3 (ab32369, Abcam, 1:100) were used as primary antibodies. All secondary antibodies Alexa 488 (Invitrogen) was diluted 1:1,000. Immunofluorescence labeling of a specific protein was used to determine its localization with the cells. Immunofluorescence images were captured using laser scanning confocal microscope (FV3000, Olympus, Japan). Left or right CeA was imaged. Integrated optical density (IOD) of the specific protein was analyzed using the Image-Pro Plus 6.0 software (Media Cybernetics, United States). The relative concentration of the labeled protein was quantified by specific thresholding of the fluorescent region of interest and by measuring the IOD of the fluorescent signal.
3
Western Blot of Zebrafish Embryo Proteins
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Western blot was performed with the standard protocols. Protein was prepared from zebrafish embryos at 44 hpf and dissolved in 4 × protein SDS–polyacrylamide gel electrophoresis Loading Buffer (Takara). Nkx2.5, Stat1a and Hdac3 were detected with anti-Nkx2.5 (PA5-49431, Thermo Fisher, US), anti-Stat1a (SAB3500364, Sigma, US) and anti-Hdac3 (ab32369, Abcam, US) at the dilution of 1:1,000, followed by incubation with an anti-rabbit IgG-horseradish peroxidase antibody (ab97069, Abcam, US) at a dilution of 1:5,000. β-actin was used as the internal control using an anti-β-actin antibody (A2228, Sigma, US), followed by an anti-mouse IgG antibody (62-6520, Invitrogen, US) diluted 1:1,000 and 1:5,000 in the block solution, respectively. Full scans of all western blots are presented in Supplementary Fig. 10 .
4
Nuclear Protein Fractionation and Western Blot
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Nuclear fraction proteins were separated using 10% SDS-PAGE with Tris-Gly buffer. Next, proteins ranging in size from 25 to 75 kDa were transferred to a 0.45 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) for 1 h at 200 mA. Proteins less than 25 kDa were transferred to a 0.22 μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) in a separate sandwich. The 0.22 µm membranes were removed 30 min after the start of the transfer. The quality of protein transfer, hybridization with primary and secondary antibodies, and protein development were assessed as described previously. For primary hybridization, rabbit antibodies (Abcam, Cambridge, UK) HDAC1 (ab7028, 1:3000) and HDAC3 (ab32369, 1:5000) were used. Goat antibodies (Abcam, Cambridge, UK, ab97051, 1:10,000) were used for secondary hybridization. To control protein loading, rabbit antibodies to histone H3 (Abcam, Cambridge, UK, ab18521, 1:3000) were used. All experiments were performed at least three times.
5
Western Blot Analysis of Histone Deacetylases
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Total protein was extracted in NP-40 lysis buffer (Boster, Wuhan, China), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto 0.45 μm PVDF membrane (Millipore). The following primary antibodies were used in the immunoblotting assays: antibodies for YY1 (ab12132, Abcam), HDAC1 (ab109411, Abcam), HDAC2 (ab32117, Abcam), HDAC3 (ab32369, Abcam) and GAPDH (AG019, Beyotime). The quantitative analysis for western blot was conducted by using Image-J Software (NIH, Bethesda, MD, USA).
6
Western Blot Analysis of HDAC3 and MMP-12
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The levels of HDAC3 and MMP-12 expressions were determined by Western blot. Liver tissue was homogenized in RIPA buffer with protease inhibitor cocktail (Sigma) on ice under the supplier's instruction. The supernatant was collected, and protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Scientific, USA). The protein was run in Mini-PROTEAN TGX Precast Gels (BioRad, USA) for polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to the nitrocellulose membrane by Semi-Dry transfer method. After keeping with 5%skim milk at room temperature for 1 h, then antibodies against HDAC3 (1:2000, ab32369, Abcam) molecular weight 49 kDa, MMP-12 (1:2000, NBP2-67344, Novus Biologicals) molecular weight 48 kDA, and actin, molecular weight 36 kDa (1:10,000, Santa Cruz Biotechnology) were added, and the membrane was incubated overnight at 4 °C. After washing with TBST, the membrane was incubated with secondary antibody, conjugated horseradish peroxidase (HRP), (goat anti-mouse and anti-rabbit, 1:10,000, Santa Cruz Biotechnology) at room temperature for 1 h. The protein bands were visualized by ECL reagent. The amount of protein levels was calculated using Bio-Rad ChemiDoc Touch Imaging System (BioRad, USA). Actin served as an internal control.
7
Investigating SFMBT2 Interactome and Modifications
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In order to confirm the binding between SFMBT2 and HDAC3 or SIAH1, or determine the acetylation level, co-IP was performed. The cells were lysed with lysis buffer (Beyotime, Shanghai, China) supplemented with 1 mM PMSF. To determine the ubiquitination level of SFMBT2, the protein extraction was performed using denature lysis buffer (containing 50 mM Tris–Cl (pH6.8) and 2% SDS). The antibody was pre-incubated with the agarose gel beads, and incubated with the lysate at room temperature for 2 h. After washing, the antigen–antibody complex was used for SDS-PAGE. After electrophoresis, transfer and blocking, the protein was incubated with the antibody against SFMBT2 (1:1000; cat no. 25256-1-AP, Proteintech, Wuhan, Hubei, China), HDAC3 (1:5000; cat no. ab32369, Abcam, Cambridge, MA, USA), SIAH1 (1:300; cat no. sc-81785, Santa Cruz, CA, USA), ubiquitin (1:5000; cat. no. NB300-130SS, Novus Biologicals, Littleton, CO, USA), flag tag (1:1000; cat. no. AE005, ABclonal, Wuhan, China), or acetylated lysine (1:300; cat. no. sc-32268, Santa Cruz, CA, USA) at 4 °C overnight. Then the protein was incubated with corresponding secondary antibody, interacted with ECL reagent, and suffered with signal exposure in the dark.
8
Immunoprecipitation of PPAR-γ, HDAC3, and RORα
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Phosphate-buffered saline was used to wash the cells before lysing them in lysis buffer (200 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 0.5% NP40). After that, the lysates were mixed with protein A beads (CST, Waltham, MA; 70024) and incubated overnight at 4°C with primary antibodies against anti-PPAR-γ (sc-7273; Santa Cruz Biotechnology, Dallas, TX), anti-HDAC3 (ab32369; Abcam, Boston, MA), anti-RORα (ab256799; Abcam), or the corresponding normal IgG (acts as negative control). To remove unbound proteins, the beads were washed twice with lysis buffer. To elute the bound proteins, the beads were resuspended in sample buffer, boiled for 3 minutes at 95°C, and immunoblotted.
9
Western Blot Analysis of Protein Expression
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The total protein was extracted according to the manufacturer’s protocols. Equal amounts of proteins (20 μg) were separated by 12% SDS-PAGE prior to transferring to PVDF membranes (Millipore, Massachusetts, USA), and then the PVDF membranes were blocked in 2% BSA at room temperature for 2 h. The membranes were incubated with primary antibodies against pan Kcr (1:1,000 dilutions; PTM Bio, PTM-502, Hangzhou, China), H2BK12cr (1:1,000 dilutions; PTM Bio, PTM-509, Hangzhou, China), H2B (1:1,000 dilutions, PTM Bio, PTM-1006, Hangzhou, China), H3 (1:1,000 dilutions, Beyotime, AF7101, Shanghai, China), HDAC1 (1:1,000 dilutions; CST, 5356, MA, USA), HDAC2 (1:1,000 dilutions; CST, 5113, MA, USA), Akt (1:1,000 dilutions, CST, 4691, MA, USA), p-Akt (Ser473) (1:1,000 dilutions, CST, 4060, MA, USA), NF-κB-p65 (1:1,000 dilutions, CST, 6956, MA, USA), β-actin (1:1,000 dilutions, CST, 4970, MA, USA), HDAC3 (1:1,000 dilutions, Abcam, ab187139, Cambridge, UK) and HDAC8 (1:1,000 dilutions, Abcam, ab32369, Cambridge, UK) at 4°C overnight. Subsequently, the membranes were detected with secondary antibodies (1:2,000 dilutions; Beyotime, Shanghai, China) at room temperature for 1 h after washing with TBST five times. Eventually, the protein signal was detected with an ECL Plus Western Blotting Detection System (Image Quant LAS 4000mini, United States).
10
Western Blot Protein Analysis Protocol
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For Western-blots, cells were lysed in RIPA buffer (#SLCD5849, Sigma, USA) and the protein concentration of each sample was determined by BCA protein assay kit (#XI357440, ThermoFisher Scientific, Waltham, USA). The protein concentration was adjusted to 20 µg of total protein which were denatured (10 min, 95oC), and applied to electrophoresis was for size fractionation (110 V, open Amp, 50 min, at 4oC) in a 4–12% SDS–PAGE (#M41212, GeneScript, Piscataway, USA). Proteins were then transferred onto a nitro-cellulose membrane (#88,018, ThermoFisher, USA) by heat-accelerated capillary transfer and over-night incubation at 50oC. Primary antibodies were applied overnight at 4oC, followed by visualization with secondary, horse radish labeled antibodies. The following antibodies were used: c-Jun (1:1000, Abcam ab40766); NF-kB p65 (1:400, Cell Signaling D14E12); GR (1:1000, Abcam ab183127); HDAC2 (1:2000, Abcam ab219053); HDAC3 (1:500, Abcam ab32369); HDAC5 (1:1000, Abcam ab55403); HDAC8 (1:1000, Invitrogen PA5-83916); GAPDH (1:1000, Abcam ab181602); α-tubulin (1:1000,R&D systems MAB9344); Anti-Rabbit IgG (1:2000, Sigma A9169-2ML); Anti-Mouse IgG (1:2000, Sigma A9917-1ML).
Blots were then quantified by the Image J (1.53 version).
Blots were then quantified by the Image J (1.53 version).
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