Proteins were extracted from HCASMCs as previously described. In brief, after rinsed with PBS, cells were harvested and proteins were extracted using radioimmunoprecipitation assay buffer. Proteins were quantified by bicinchoninic acid assay after sonication and centrifugation. They were loaded in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (6–9%, 5–10 μg per lane). Antibodies Osteopontin (abcam, 1:500), Collagen III (abcam, 1:500), α-SMA (abcam, 1:500), Calponin 1 (abcam, 1:500), HDAC3 (abcam, 1:500), WDR5 (abcam, 1:1000), NADPH (abcam, 1:1000), NOX1 (abcam, 1:1000), GAPDH (abcam, 1:2000) were used in this study. Images were collected by ImageQuant LAS 4000 Imaging Station (GE), the densities of bands were quantified with the ImageQuant TL software (GE).
Hdac3
Manufactured by Abcam
Sourced in United States, United Kingdom
HDAC3 is a histone deacetylase enzyme that is involved in the regulation of gene expression. It plays a role in the deacetylation of histones, which can lead to changes in chromatin structure and gene transcription.
Automatically generated - may contain errors
Lab products found in correlation
Hdac2, by Abcam (8 mentions)
Hdac1, by Abcam (7 mentions)
Gapdh, by Abcam (3 mentions)
Ecl plus reagent, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Bioworld Technology (2 mentions)
Vinculin, by Merck Group (2 mentions)
V5 tag, by Thermo Fisher Scientific (2 mentions)
H3k4me1, by Abcam (2 mentions)
Hdac3, by GeneTex (2 mentions)
Acetylated lysine rabbit polyclonal, by Cell Signaling Technology (2 mentions)
Ha tag, by GeneTex (2 mentions)
Myc tag, by GeneTex (2 mentions)
Pol 2 antibody n 20, by Santa Cruz Biotechnology (2 mentions)
Gcn5 h 75, by Santa Cruz Biotechnology (2 mentions)
Mouse monoclonal sb62a anti rabbit igg light chain hrp, by Abcam (2 mentions)
Hsp90, by Cell Signaling Technology (2 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (2 mentions)
Anti mouse igg hrp, by Cell Signaling Technology (2 mentions)
β actin, by Abcam (2 mentions)
H3k27ac, by Abcam (2 mentions)
27 protocols using hdac3
1
Quantitative Protein Analysis of HCASMCs
2
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with 0.5% NP40 lysis buffer and western blotting was performed according to the standard protocol. Detection was accomplished with the chemiluminescence ECL plus reagent (Thermo, Grand Island, NY, USA) and the chemiluminescence HRP substrate (Millipore, Billerica, MA, USA), and signals were evaluated by a Tanon 5200Multi scanner (Shanghai, China). Primary antibodies were as follows: HDAC1 (Abcam, Cambridge, UK), HDAC2 (Abcam), HDAC3 (Abcam), cleaved caspase-3 (CST, Danvers, MA, USA), cleaved PARP (CST), PARP (CST), GAPDH (Bioworld, St. Louis Park, MN, USA), K9 acetyl-histone H3 (CST), Bax (CST), P53 (Santa Cruz), PUMA (CST), and HA (CST).
3
Immunoblot Analysis of Apoptosis Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblots were carried out as described by our group [23 ,24 (link)]. Original immunoblots see File S1. Antibodies used for this assay were: BCL-XL (#ab32370), survivin (#ab134170), p21 (#ab109520), BAX (#ab32503), BAK (#ab32371), BIM (#ab32158), HDAC3 (#ab32369), GAPDH (#ab128915) from Abcam, Cambridge, UK; MCL-1 (#sc-819), HDAC8 (#sc-374180), HSP90 (#sc-13119), vinculin (#sc-73614) from Santa Cruz Biotechnology, Heidelberg, Germany; cleaved caspase-3 (#cs9661), PARP1 (#cs9542), BID (#cs2002), HDAC1 (#cs34589), HDAC2 (#cs5113), histone H3 (#cs14269), ac-histone H3 (K9) (#cs9649), ac-histone H3 (K18) (#cs9675), ac-histone H3 (K27) (#cs8173) from Cell Signaling, Leiden, The Netherlands; ac-tubulin (#T7451) from Sigma-Aldrich, Taufkirchen, Germany; ac-histone H3 (#06-599) from Merck Millipore, Burlington, MA, USA; and NOXA (#ALX-804-408) from Enzo Life Sciences, New York, NY, USA. HSP90, GAPDH, and vinculin served as independent housekeeping proteins to normalize protein loading. The protein ladders used were the PageRulerTM pre-stained protein ladder (#26616) and the PageRulerTM Plus pre-stained protein ladder (#26619) from Thermo Fischer Scientific, Waltham, MA, USA.
4
Comprehensive Antibody Panel for Western Blotting and ChIP
5
Western Blot Analysis of Epigenetic Regulators
6
Immunohistochemical Analysis of HDAC3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sections (3-μm) were dewaxed and hydrated which was followed by antigen retrieval by high-temperature and high-pressure for 3 min. The sections were blocked with 3% peroxidase and probed with the primary antibody HDAC3 (1:2000, Abcam Inc., Cambridge, MA, USA) as well as the secondary antibody. After that, the sections were developed by diaminobenzidine (DAB), which was followed by counterstaining with hematoxylin solution and sealing. A negative control (NC) was set with phosphate buffered saline (PBS) as the primary antibody. Streptavidin-peroxidase staining kit and DAB kit were provided by Beijing Zhongshan Biotechnology Co. Ltd. (Beijing, China). HDAC3 is mainly located in the nucleus [20 (link)] and the positive staining shows in brownish yellow. Each sections were evaluated by National Institutes of Health ImageJ software in 5 high-power fields. The ratio of positive cells to total cells in each field was calculated.
7
Autophagy regulation by KLF5 and Bcl2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3-methyladenine (3-MA, 189490) and Bafilomycin A1 (BAFA1, 196000) were purchased from EMD Millipore (Darmstadt, Germany). Docetaxel and PF4708671 were from Selleck Chemicals (Houston, TX, USA). Lipofectamine 2000 reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RIPA buffer was acquired from Cell Signaling Technology (Danvers, MA, USA), protease inhibitor and phosphatase inhibitor were from Abcam (Cambridge, MA, USA). Roche (Mannheim, Germany). Primary antibodies against LC-3 I/II, Beclin1, ATG3, ATG5, ATG7, mTOR, p-mTOR (Ser2448), ACC, p-ACC (Ser79), p-AMPK(Thr172), and AMPK were acquired from Cell Signaling Technology and HDAC3 was from Abcam (Cambridge, MA, USA). PVDF membrane was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The siKLF5 #1, siKLF5 #2 siRNAs, siBeclin1 #1, siBeclin1 #2 siRNAs, siBcl2 #1, siBcl2 #2 siRNAs and the scrambled RNA, which used as shcontrol were obtained from RiboBio (Guangzhou, China). PLKO.1 lentiviral vectors encoding short hairpin RNA (shRNA) targeting non-specific control (NC) or human KLF5 (sh710: 5′-GGTTACCTTACAGTATCAACA-3′) were constructed by GenPharma (Shanghai, China).
8
Histone Deacetylase and Inflammasome Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spinal cord tissue and primary neurons were lysed by lysis buffer (Beyotime, Shanghai, China). After protein quantification, electrophoresis (12% SDS-PAGE gels), and Western transfer, the membranes were blocked with 5% BSA and incubated with the appropriate primary antibodies, such as HDAC1 (CST, 1:1,000), HDAC2 (Abcam, 1:800), HDAC3 (Abcam, 1:1,000), NLRP3 (Thermo Fisher Scientific, 1:1,000), caspase-1 (Proteintech, 1:800), and caspase-1 p20 (CST, 1:1,000) at 4°C overnight and then incubated with secondary antibodies (Abgent, 1: 20,000) followed by the hypersensitivity chemiluminescent substrate (Bio-Rad, Hercules, CA, United States), and protein bands were photographed by using a ChemiDoc™ MP imaging system (Bio-Rad).
9
Protein Expression Analysis in Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using RIPA buffer (Santa Cruz Biotechnology, Inc.) supplemented with a protease inhibitor cocktail (Roche Applied Science). Protein concentration was determined using a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Next, 30 µg total protein from each lysate was separated using 10 or 12% SDS-PAGE and subsequently electroblotted onto a PVDF membrane. The membranes were blocked with 5% skimmed milk at room temperature for 2 h. The target proteins were probed with primary anti-bodies (1:1,000) overnight at 4°C and further incubated with HRP-conjugated secondary antibody (cat. no. ab7090; Abcam; 1:10,000). The primary antibodies were specific for GAPDH (cat. no. ab181602; Abcam), HDAC1 (cat. no. ab53091; Abcam), HDAC2 (cat. no. ab7029; Abcam), HDAC3 (cat. no. ab96005; Abcam), HDAC8 (cat. no. 17548-1-AP; ProteinTech Group, Inc.), MMP-2 (cat. no. ab97779; Abcam), MMP-9 (cat. no. PA5-27191; Thermo Fisher Scientific, Inc.), E-cad (cat. no. ab231303; Abcam) and Slug (cat. no. ab27568; Abcam). GAPDH was used as the loading control for normalization. The signals were detected by enhanced chemiluminescence using a Chemidoc XRS Molecular Imager (Bio-Rad Laboratories Inc.). Quantity One software (version 4.3.0; Bio-Rad Laboratories, Inc.) was used for densitometric analysis.
10
Hdac3 Immunohistochemistry in Tumors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!