ChIP assay kits (Millipore) were used as follows: A total of 1 × 107 cells were fixed with 1% formalin for 10 min to make DNA and protein cross-linked. After that, the cells were subjected to SDS lysis, and the DNA was randomly sonicated into 500–1000 bp fragments. After 12,000 g centrifugation at 4 ℃, the supernatant was collected and placed into two tubes, which were respectively mixed with the specific antibodies of the target proteins. Pol II (05–623, 10 mL/mg total protein, Millipore), AcH4 (06-866, 10 mL/mg total protein, Millipore), HDAC2 (17-10237, 5 mL/mg total protein, Millipore) and IgG in the control group. For AcH4 ChIP, sodium butyrate (20 mM, 19-137, Millipore) was added to all solutions to maintain histone acetylation. ChIP DNA was purified and eluted with 100 μL H2O, and 2.5 μL ChIP DNA was extracted for qPCR detection. Primers: RNA Pol II: 5ʹ-CAGGGACTGGGAGAAGGA-3ʹ (forward), 5ʹ-CACTGCTAGTGACAGGTGCA-3ʹ (reverse); AcH4 and HDAC2: 5ʹ-GCCCCATCCCCTGCTGCT-3ʹ (forward), 5ʹ-CAGGCCCAGCGACTCACC-3ʹ (reverse).
Hdac2
Manufactured by Merck Group
Sourced in Canada
HDAC2 is a histone deacetylase enzyme that is involved in the regulation of gene expression. It is responsible for the removal of acetyl groups from the lysine residues of histone proteins, which can lead to chromatin compaction and transcriptional repression.
Automatically generated - may contain errors
Lab products found in correlation
Nuclear complex co ip kit, by Active Motif (3 mentions)
Histone h4, by Cell Signaling Technology (2 mentions)
Hrp conjugated anti mouse igg, by Avantor (2 mentions)
Donkey anti rabbit igg, by Avantor (2 mentions)
Chip assay kit, by Merck Group (1 mentions)
Fibronectin, by Santa Cruz Biotechnology (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Histone h3, by Merck Group (1 mentions)
Polyvinyl difluoride membrane, by Merck Group (1 mentions)
Histone h4, by Merck Group (1 mentions)
Ac histone h3, by Merck Group (1 mentions)
α sma, by Santa Cruz Biotechnology (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated donkey anti rabbit igg, by GE Healthcare (1 mentions)
H3k36me2, by Merck Group (1 mentions)
H3k27me3, by Merck Group (1 mentions)
C myc, by Abcam (1 mentions)
Hcfc1, by Merck Group (1 mentions)
10 protocols using hdac2
1
ChIP Assay for Histone and Transcription Factor
2
Western Blot Analysis of EMT Markers
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A549 cells were lysed in PRO-PREPTM protein extraction solution (iNtRON Biotechnology, Seongnam, Korea). Lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinyl difluoride membranes (Millipore Inc., Billerica, MA). Membranes were blocked with a 5% skim milk solution and incubated with the following antibodies: E-cadherin, vimentin, α-SMA, fibronectin, snail, slug (Santa Cruz, CA), HDAC2, HDAC4, ac-histone H3, histone H3, ac-histone H4, histone H4 (Upstate, Millipore Inc.), and β-actin (Santa Cruz, CA). The blots were visualized with HRP-conjugated secondary antibodies and an ECL system (Pierce, Rockford, IL).
3
Epigenetic Protein Complex Profiling
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Nuclear extracts were prepared using the Nuclear Complex Co-IP kit (Active Motif). Proteins were separated using SDS-PAGE, transferred to PVDF membranes, and probed with antibodies to EZH2 (5246, Millipore), HDAC2 (05–814, Millipore), NSD2 (39 (link)), and histone H4 (2935, Cell Signaling Tech.). Secondary antibodies were HRP-conjugated anti-mouse IgG (95017–332, VWR) or donkey anti-rabbit IgG (95017–556, VWR).
4
Nuclear and Cytoplasmic Fractionation
5
Chromatin Immunoprecipitation of HDACs and Histone Modifications
6
Nuclear Protein Extraction and Detection
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Nuclear proteins were extracted using the Nuclear Complex Co-IP Kit (Active Motif). Proteins were electrophoretically separated, blotted and detected using enhanced chemiluminescence. Primary antibodies used were: H3K36me2 (Millipore 07-369), H3K27me3 (Millipore 07-449), MMSET [12] (link), c-MYC (Abcam ab32072), HDAC2 (Millipore 05-814) and pan-H4 (Abcam ab7311). The secondary antibody used was horseradish peroxidase-conjugated donkey anti-rabbit IgG (GE Healthcare Life Sciences).
7
Epigenetic Protein Complex Profiling
8
Cloning and Mutagenesis of OTUD5 Variants
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pDEST-FLAG-HA-USP5 and pDEST-FLAG-HA-OTUD5 were gifts from W. Harper (Addgene plasmids #22590 and #22610) (75 (link)). pRK5-HA-Ub-K48only and pRK5-HA-Ub-K63only were gifts from T. Dawson (Addgene plasmids #17605 and #1706). OTUD5 patient mutations (G494S, L352P, 161-164del, R274W, and D256N), point mutations (C224S or C224R), truncation mutations (ΔCterm = OTUD5 1-534, Cterm = OTUD5 534-571), and wobble mutations to make constructs resistant to shOTUD5#5 were introduced in this vector using the Q5 site-directed mutagenesis kit (E0554, NEB) following the manufacturer’s instructions. OTUD5 and OTUD5C224R were further subcloned into pKmyc using Bam HI and Not I sites (pKmyc-OTUD5 WT or C224R). For expression in hESCs, OTUD5 variants were cloned into pENTR1A or pENTR233 and recombined into pINDUCER20 (76 (link)). pLKO1-Puro Mission shRNA constructs targeting OTUD5 (#2: TRCN0000122275 and #5: TRCN0000233196), ARID1A (TRCN0000059092), ARID1B (TRCN00000420576), UBR5 (TRCN0000003411), HDAC2 (TRCN00000004819), HCFC1 (TRCN00000001625), TRAF3 (TRC00000034219), and TRIM25 (TRC0000003499) were purchased from Sigma-Aldrich. siRNA pools were purchased from Santa Cruz Biotechnology and Thermo Fisher Scientific.
9
Methionine Deprivation Modulates HDAC2 in Lung Metastasis
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Six-week-old BALB/c nude mice were provided by Nanchang University’s Laboratory Animal
Science Center (Nanchang, China). All experiments were approved by the Institutional
Animal Care and Use Committee of the Second Affiliated Hospital of Nanchang University.
MKN45 cells (2×10 6 cells) were injected into BALB/c nude mice via the caudal
vein. After injection, mice were divided into the Met + group (
n = 6) and the Met – group ( n = 6). Mice
in the Met + group were given a normal diet, while mice in the Met ‒group were given a methionine-free diet. Eight weeks later, the mice were sacrificed, and
lung tissue was collected.
MKN45 cells (2×10 6 cells) transfected with lentivirus-sh-HDAC2 or
lentivirus-sh-NC (RiboBio) were injected into BALB/c nude mice via the caudal vein. After
injection, mice were split into four groups: Met ++sh-NC, Met
++sh-HDAC2, Met ‒+sh-NC, and Met ‒+sh-HDAC2. Mice in the
Met + group were given a normal diet, while mice in the Met – group
were given a methionine-free diet. Eight weeks later, the mice were sacrificed, and the
lung tissue was collected. The target sequence of shRNA HDAC2 (TRCN0000004823; Sigma) is:
5′-GCAAATACTATGCTGTCAATT-3′. The control (sh-NC, 5′-GACCTGTACGCCAACACAGTG-3′; RiboBio) is
a meaningless sequence attached to the HDAC2 shRNA that does not target the human genome.
Science Center (Nanchang, China). All experiments were approved by the Institutional
Animal Care and Use Committee of the Second Affiliated Hospital of Nanchang University.
MKN45 cells (2×10 6 cells) were injected into BALB/c nude mice via the caudal
vein. After injection, mice were divided into the Met + group (
n = 6) and the Met – group ( n = 6). Mice
in the Met + group were given a normal diet, while mice in the Met ‒group were given a methionine-free diet. Eight weeks later, the mice were sacrificed, and
lung tissue was collected.
MKN45 cells (2×10 6 cells) transfected with lentivirus-sh-HDAC2 or
lentivirus-sh-NC (RiboBio) were injected into BALB/c nude mice via the caudal vein. After
injection, mice were split into four groups: Met ++sh-NC, Met
++sh-HDAC2, Met ‒+sh-NC, and Met ‒+sh-HDAC2. Mice in the
Met + group were given a normal diet, while mice in the Met – group
were given a methionine-free diet. Eight weeks later, the mice were sacrificed, and the
lung tissue was collected. The target sequence of shRNA HDAC2 (TRCN0000004823; Sigma) is:
5′-GCAAATACTATGCTGTCAATT-3′. The control (sh-NC, 5′-GACCTGTACGCCAACACAGTG-3′; RiboBio) is
a meaningless sequence attached to the HDAC2 shRNA that does not target the human genome.
10
Chromatin Remodeling Protein Analysis
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