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).
Hdac2
Manufactured by Abcam
Sourced in United Kingdom, United States
HDAC2 is a protein that functions as a histone deacetylase, which plays a role in the regulation of gene expression. It is involved in the deacetylation of histones, a process that can lead to changes in chromatin structure and gene silencing.
Automatically generated - may contain errors
Lab products found in correlation
Hdac1, by Abcam (16 mentions)
Hdac3, by Abcam (8 mentions)
β actin, by Merck Group (5 mentions)
Ecl plus reagent, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Abcam (3 mentions)
Gapdh, by Bioworld Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
α tubulin, by Merck Group (2 mentions)
Pan acetyl h4, by Merck Group (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Foxk2, by Fortis Life Sciences (2 mentions)
Foxk1, by Santa Cruz Biotechnology (2 mentions)
Sin3a58, by Santa Cruz Biotechnology (2 mentions)
Sin3b, by Santa Cruz Biotechnology (2 mentions)
Pf1 phf12, by Cell Signaling Technology (2 mentions)
Mrg15, by Cell Signaling Technology (2 mentions)
Foxo343, by Merck Group (2 mentions)
Hdac8, by Abcam (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
C myc, by Abcam (2 mentions)
39 protocols using hdac2
1
Western Blot Analysis of Apoptosis Markers
2
Protein Expression Analysis of Cardiac Remodeling
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Scar and LV tissue were dissected from AMI, CHF, and sham hearts, respectively. Dissected tissues and isolated cells were homogenized in lysis buffer (50 mM Tris- HCl pH7.5, 150 mM NaCl, 0.5% NP-40 (Sigma), 0.5% Triton-X (Sigma), 1 mM EDTA (Sigma), and complete mini protease inhibitor (Roche). Protein concentrations were determined by BCA assay (Thermo Scientific). Typically, 40 μg of protein was loaded on 4% to 12% Tris-Bis gels (Life Technologies), separated in MOPS running buffer, and transferred to a PVDF membrane (Millipore). After blocking with 5% non-fat dry milk in 1× TBS, membranes were probed with HDAC1, HDAC2 (Abcam, 1:1,000), α-SMA (Sigma, 1:5,000), E-cadherin, GSK3β (Santa Cruz, 1:500), p-GSK3β, Cleaved Caspase 3, p-Akt, Akt (Cell Signaling, 1:1,000), β-Catenin active (Millipore,1:1,000), and β-Actin (Sigma, 1:50,000) in TBS with 5% milk overnight at 4°C. Following three washes in TBS, membranes were incubated with HRP-conjugated secondary antibodies (Santa Cruz, 1:50,000) for 1 h at room temperature. An ECL (Millipore) system was used for detection of the bands and exposed to X-ray film (Thermo Scientific) in a dark room. Densitometry analysis was performed with Alpha Ease FC software.
3
Antibody Validation for Protein Analysis
4
Quantifying Collagen 1 Expression in Kidney
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Protein lysates were made by homogenizing kidney tissue in cold 50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X‐100, and 200 mM sucrose with protease inhibitors using a Tissue Tearor. Protein lysates were run on SDS‐PAGE gels and proteins detected using Collagen 1 (Proteintech, cat# 14695‐1‐AP, 1:3,000) and HDAC2 (Abcam, cat# ab32117, 1:3,000) primary antibodies and peroxidase‐labeled goat anti‐rabbit secondary antibody (Sigma‐Aldrich, cat# A0545, 1:10,000). Signal was developed with SuperSignal West Dura (Thermo Fisher). Collagen 1 densitometry was performed using Image J and normalized to HDAC2.
5
CUT&RUN Profiling of Histone Deacetylases in Forelimb Development
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Experiments were performed according to EpiCypher’s CUTANA CUT&RUN protocol (EpiCypher #15-1016) with the following modifications. 200,000–300,000 cells from E9.25 (21–23 S) forelimbs and anterior E10.5 (32–35 S) forelimbs were incubated overnight at 4 °C on a nutator with 1:250 FLAG primary antibody (Sigma #F3165), 1:100 HDAC1 (Abcam ab7028) or 1:200 HDAC2 (Abcam ab7029). The following day, samples were incubated at room temperature for 30 min. with secondary antibody, 1:100 Donkey anti-mouse (Jackson ImmunoResearch #715-035-150) or Donkey anti-rabbit (Jackson ImmunoResearch #711-005-152), followed by three washes in Digitonin wash buffer. CUTANA pAG-MNase was then incubated with samples for 10 min at room temperature and then the MNase reaction was performed for 2 h at 4 °C on a nutator. Libraries were generated using NEBNext Ultra II DNA Library Prep Kit with 14 PCR cycles and cleaned up to remove adapters using AMPure XP beads (Beckman Coulter) or SparQ PureMag beads (QuantaBio). Samples were sequenced on an Illumina NextSeq 500 instrument using PEx75 to a depth of depth of 3–5 million reads. Peaks were called using MACS79 (link).
6
Antibody Validation for Protein Analysis
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Antibodies used were FOXK1 (Santa Cruz sc-134550, 1:2000), FOXK2 (Bethyl A301-730A-1, 1:1000), Sin3A58 (link) (1:5000), SIN3B (Santa Cruz sc-768, 1:1000), HDAC2 (Abcam ab7029, 1:10000), SAP3059 (link) (1:1000), Sds3/SAP4560 (link) (1:1000), Pf1/Phf12 (Bethyl A301-647A, 1:1000), RBP2/Kdm5a61 (link) (1:1000), MRG15 (1:1000), LC3B (Cell Signaling #2775, 1:2000), p62/Sqstm1 (MBL PM045, 1:5000), α-tubulin (Sigma #T5168, 1:5000), H2B (Abcam ab1790, 1:2000), H4 (Millipore 05-858), pan-acetyl H4 (Millipore 06-866), FOXO343 (link), and Flag (Sigma #F7425, 1:500). The specificities of the FOXK1 and FOXK2 antibodies were validated by their specific recognition of their intended antigens in co-immunoprecipitation experiments (Fig. 1c , Supplementary Figure 1 ), as well as in lysates from cells depleted of either FOXK1 or FOXK2 (Fig. 1d ).
7
Molecular Analysis of Prefrontal Cortex
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Samples from mice ACC were collected. Total proteins were lysed by M-PER Protein Extraction Buffer, and nuclear proteins of ACC were extracted with nuclear extraction kit (Thermo) according to the manufacturer’s instructions. Protein concentrations were determined using a BCA Kit. Equal amounts of protein aliquots were used for Western blot analysis to check the expression levels of LXRα, LXRβ, p50, p65, HDAC2, HDAC5, Iba-1 (Abcam), phosphorylated IκBα (p-IκBα, Bioworld Technology), phosphorylated extracellular regulated protein kinases (p-ERKs), ERK, phosphorylated p38 (p-p38), p38, phosphorylated c-Jun N-terminal kinase (p-JNK), JNK, GFAP (Cell Signaling Technology), AcH3 and AcH4 (Millipore), Histone H3, Histone H4, β-tubulin III (Proteintech), and β-actin (Sigma-Aldrich) served as a loading control.
8
Histone Peptide Pull-Down Assay
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One hundred micrograms of lyophilized histone peptides (H3K4ac, Epicypher; unmodified H3, H3K4me1, H3K4me2, H3K4me3; Millipore) were dissolved in 400 µL of PBS. 1 × 108 MEFs were hypotonically lysed with Buffer A (10 mM HEPES at pH 7.9, 1.5 mM MgCl2, 10 mM KCl, freshly added protease inhibitors and DTT to 1 mM) before nuclear extract was isolated with Buffer C (20 mM HEPES at pH 7.9, 25% v/v glycerol, 420 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, freshly added protease inhibitors and DTT to 1 mM) and the salt concentration lowered to 150 mM using Buffer D (20 mM HEPES at pH 7.9, 20% v/v glycerol, 0.2 mM EDTA, 0.2% Triton X-100). Following mechanical lysis by sonication, 50 µL of prewashed avidin-histone peptide slurry was added to precleared lysate and incubated overnight at 4°C. After washing of the avidin beads, SDS sample buffer was added and samples were boiled for 5 min and subsequently run on a 10% SDS gel. The following antibodies were used for the histone peptide pull-down assay: mSin3A (Abcam, ab3479), CoREST (Millipore, 07–455), HDAC2 (Abcam, ab7029), HDAC1 (Abcam, ab7028), RBBP4 (Abcam, ab79416), MTA1 (Santa Cruz, sc-9446).
9
Chromatin Immunoprecipitation of HDAC2 and AcH3K9/14
10
HDAC2 Enzymatic Activity Assay
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