Protein levels were measured by immunoblot analysis following a previous protocol 26 (link). Tissues and cells were lysed in 1×RIPA lysis buffer (Abcam, #ab156034). After boiling with Laemmli SDS sample buffer (Sigma-Aldrich, #S3401), equal amount of total cell lysate of each sample was loaded onto 10% SDS-PAGE gels, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, #LC2002). The immunoblots were then probed with primary antibodies including anti-HDAC1 (Abcam, #ab53091), anti-FOXP3 (Abcam, #ab450), anti-CtBP2 (BD Biosciences, San Jose, CA, USA, #612044), anti-NLRP1 (Biolegend, San Diego, CA, USA, #a679802), anti-Caspase-1 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-56036), and anti-GAPDH (Santa Cruz Biotechnology, #47724). Protein signals were recorded using enhanced chemiluminescence reagents (GE Healthcare Life Sciences, Piscataway, NJ, USA, #RPN2232).
Ab53091
Manufactured by Abcam
Sourced in United States
Ab53091 is a laboratory equipment product manufactured by Abcam. It is a device designed for laboratory use, but no further details about its core function can be provided without the risk of extrapolation or unintended interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Anti ctbp2, by BD (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 1, by Santa Cruz Biotechnology (1 mentions)
Laemmli sds sample buffer, by Merck Group (1 mentions)
Anti foxp3, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab10158, by Abcam (1 mentions)
Nuclear extraction and fractionation protocol, by Abcam (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Triethylamine, by Merck Group (1 mentions)
Folic acid, by Merck Group (1 mentions)
N hydroxysuccinimide, by Merck Group (1 mentions)
Lipoic acid, by Merck Group (1 mentions)
Poly allylamine hydrochloride pah, by Merck Group (1 mentions)
N n dicyclohexylcarbodiimide, by Merck Group (1 mentions)
Ab55391, by Abcam (1 mentions)
10 protocols using ab53091
1
Quantifying Protein Levels by Immunoblotting
2
Evaluating HDAC Expression in HeLa Cells
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The effect of LCS-1269 on HDAC expression was assessed on HeLa TI cells using Western blotting. Cells were incubated with LCS-1269 at concentrations of 2 and 4 μM for 24 h in 60 mm Petri dishes. The nuclear fraction of the cells was obtained according to the Abcam Nuclear extraction and fractionation protocol. Nuclear proteins were separated by 10% PAGE and transferred to 0.22 μm nitrocellulose membranes; transfer conditions for proteins up to 25 kDa were 40 min at 100 mA, for proteins from 25 to 100 kDa—40 min at 100 mA and 20 min at 200 mA. Blocking with BSA, primary and secondary antibodies was performed as described above. The study used Abcam antibodies to HDAC1 (ab53091), histone H4 (load control, ab10158) and secondary anti-rabbit antibodies (ab6721). Protein detection and densitometric analysis were performed as described above. Experiments were performed three times.
3
Functionalization of MoS2 Nanosheets
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We purchased monolayer MoS2 nanosheets (62 μg mL-1, dissolved in ethanol) from 2D-Semiconductors. mPEG-NH2 (5K) and NH2-PEG-Boc (5K) from Laysan Bio, Inc (USA). We purchased Lipoic acid (LA), Folic acid (FA), trifluoroacetic acid (TFA), dichloromethane, N,N'-dicyclohexylcarbodiimide (DCC), triethylamine (TEA), sodium bicarbonate, N-Hydroxysuccinimide (NHS), dimethyl sulfoxide (DMSO), diethyl ether, Poly(allylamine hydrochloride) (PAH, molecular weight 15,000), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromides (MTT) from Sigma Aldrich. Antibodies against HDAC1 (ab53091), KRAS (ab55391), and Actin (ab8227) were obtained from Abcam. We used ultrapure DI water for conducting all experiments (Milli-Q Integral 5).
4
Investigating HDAC1 and IKZF1 Interaction
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The interaction between HDAC1 and IKZF1 proteins was verified as follows. The 293 T cells (CL-0005, Procell, culture conditions were the same as RAW 264.7 cells) were transfected with oe-NC, oe-HDAC1, oe-IKZF1 or co-transfected with oe-HDAC1 and oe-IKZF1 employing Lipofectamine 2000 reagent (12566014, Thermo Fisher Scientific) for 48 h. Next, cells were lysed with 300 μL cell lysate containing protease inhibitor. The supernatant was collected, 50 μL of which served as Input, and the remaining was incubated with 2 μg antibody to IKZF1 (#5443, 1: 50, Cell Signaling Technologies [CST], Beverly, MA) or IgG (#3423, 1: 20, CST) overnight at 4 °C. Thereafter, the sample was added with 20 μL protein A/G-sepharose microspheres (36403ES03, Yeasen Company, Shanghai, China), and shaken for 3 h at 4 °C. The cells were collected, washed with cell lysate 3 times, heated in 40 μL loading buffer and precipitated for 5 min. Western blot analysis was implemented to quantify the protein expression of HDAC1 (ab53091, 1: 1000, Abcam) and IKZF1 (#5443, 1: 50, CST). The interaction between HDAC1 and IKZF1 proteins in RAW 264.7 cells was detected with the same procedures and same antibodies as the above. The cells were grouped into control, LPS, LPS + PU, LPS + oe-NC, and LPS + oe-HDAC1 groups.
5
Protein Expression Analysis of HDAC1, PP2A, NLRP3, and Caspase-1
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Total protein was extracted, electrophoresed and then electroblotted to a polyvinylidene fluoride membrane which was incubated with primary antibodies against HDAC1 (ab53091, 1: 1000, Abcam), PP2A (ab32065, 1: 500, Abcam), NLRP3 (ab263899, 1: 1000, Abcam), pro-IL-1β (ab234437, 1: 1000, Abcam), Caspase-1 (ab138483, 1: 1000, Abcam), Caspase-1 p20 (sc-398715, 1: 2000, Santa Cruz Biotechnology, CA) and β-actin (ab8226, 1: 5000, Abcam, internal reference) at 4 °C overnight. Horseradish peroxidase-labeled secondary antibody of goat anti-rabbit IgG (ab97051, 1: 2000, Abcam) was added for another 1-h of culturing with membrane. The membrane was immersed in an enhanced chemiluminescence reaction solution (BM101, Biomiga) for development.
6
ChIP Assay Procedure for Protein-DNA Interactions
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The ChIP assay procedure was performed as described previously 29 , 30 (link). In brief, cells were primarily washed with phosphate buffered saline (PBS) buffer, followed by crosslinking with 1% formaldehyde (Polysciences, Inc. Warrington, PA, USA, #18814) at 37°C for 15 min. The fixed cells were then subjected to a standard ChIP assay using a high-sensitivity ChIP kit (Abcam, #ab185913) following the manufacturer's instructions. The antibodies used in this assay included anti-HDAC1 (Abcam, #ab53091), anti-FOXP3 (Abcam, #ab450), and anti-CtBP2 (BD Biosciences, #612044). The enriched DNA was then subjected to qRT-PCR analyses using the primers listed in Supplementary Table-3.
7
Western Blot Analysis of Lipid Regulatory Proteins
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The tissues and cultured cells were lysed by RIPA buffer (Beyotime) containing 0.1 mmol/L phenylmethylsulfonyl fluoride. Protein extracts were quantified and then subjected to SDS-PAGE, followed by immunoblotting with mouse monoclonal antibody against ABCA1 (ab18180, 1:500, Abcam, Cambridge, MA, USA), rabbit monoclonal antibody against ABCG1 (ab52617, 1:1000, Abcam), rabbit monoclonal antibody against CD36 (ab133625, 1:500, Abcam), mouse polyclonal antibody against SR-A (AF1797, 1:1000, R&D Systems, MN, USA), rabbit monoclonal antibody against LXRα (ab176323, 1:500, Abcam), rabbit polyclonal antibody against HDAC1 (ab53091, 1:1000, Abcam), rabbit monoclonal antibody against HDAC3 (ab32369, 1:1000, Abcam), mouse monoclonal antibody against HDAC5 (ab50001, 1:300, Abcam), mouse monoclonal antibody against HAT-1 (sc-390562, 1:500, Santa Cruz, TX, USA), or rabbit monoclonal antibody against β-actin (ab115777, 1:1000, Abcam). After rinsing with PBS-T, the membranes were incubated with HRP-labeled secondary antibodies (1:3000, Beyotime). The proteins were visualized using Tanon 5500 (Shanghai, China) and BeyoECL Plus (Beyotime), and β-actin was used as an internal control.
8
Characterizing Transcriptional Regulators
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Anti-Flag (205431AP, Proteintech: dilution 1:3000 for western), -Six2 (66347, Proteintech: dilution 1:150 for IHC), -Rest (ABIN747683, Antibodies-online: dilution 1:100 for IHC and 1:2000 for western), -Cdyl (17763–1-AP, ThermoFisher: dilution 1:200 for IHC and 1:1000 for western), and -HDAC1 (ab53091, Abcam: dilution 1:2000 for western), and -Hltf (MBP183256, Novus Biologicals: dilution 1:100 for IHC and 1:2000 for western; MABE1074, Millipore-Sigma: dilution 1:60 for IHC).
9
Western Blot Analysis of Apoptosis and NF-kB Signaling
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The cell lysates of SiHa and HeLa cells were prepared using RIPA lysis buffer (Beyotime, China) and then measured by Bradford Protein Assay kit (Bio-Rad, USA). After denaturation at 95°C, protein samples penetrated through SDS-polyacrylamide gel and were then electrotransferred to polyvinylidenedifluoride membranes (Bio-Rad). Afterwards, the membranes were blocked with Tris-buffered saline containing 5% non-fat dry milk for 1 hr at room temperature before being mixed with the primary antibodies overnight at 4°C. After being washed by PBS, HRP-conjugated secondary antibodies (1:5000, #ab205718, Abcam, Cambridge, UK) were cultured with membranes at 4°C for 1 hr. Protein bands were detected with an enhanced chemiluminescence detection system (Millipore, Billerica, MA, USA). GAPDH served as the internal control and nucleoproteins were normalized by HDAC1 gene. Antibodies used in this study were as follows: cleavedcaspase-3 (1:500, #ab13847, Abcam), B-cell lymphoma 2 (Bcl-2, 1:1000, #ab32124, Abcam), Bax (1:1000, #ab32503, Abcam), TLR4 (1:500, #ab13556, Abcam), p-P65 (1:1000, #ab86299, Abcam), t-P65 (1:1000, #ab237591,Abcam) and Histone deacetylase 1 (HDAC1,1:300, #ab53091, Abcam).
10
Moscatilin Regulates HDAC Expression
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Forty-eight hours after the moscatilin treatment, MDA-MB-231 cells were lysed using the RIPA lysis buffer (50 mM Tris (pH7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktails). The protein expression levels of indicated genes were detected using Western blotting as previous described.18 (link) The primary antibody of HDAC1 (ab53091, 1:1500 dilution), HDAC2 (ab137364, 1:1000 dilution), and GAPDH (ab181602, 1:2000 dilution) were purchased from Abcam (Cambridge, UK); HDAC3 (#3949, 1:1000 dilution), H3K9Ac (#9677, 1:1000 dilution), H3K9Ac (#13534, 1:1000 dilution), H3 (#9728, 1:2000 dilution), and H4 (#13944, 1:2000 dilution) were obtained from Cell Signaling Technology, Inc. (Danvers, USA).
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