For RNA pull‐down, the procedure was performed with minor modifications.20 Briefly, biotin‐labeled RP11‐367G18.1 variant 2 was synthesis by in vitro transcription and then purified. The biotinylated RP11‐367G18.1 variant 2 was incubated with cell lysates at 4°C for 6 h. The streptavidin agarose beads were added into the mixture and incubated for 1 h. The beads were washed and boiled for western blot analysis. As previously described,18 proteins and histones extracted from the cells were analyzed by SDS–PAGE using antibodies against HIF‐1α, N‐cadherin (BD Biosciences), H4K5Ac, H4K8Ac, H4K12Ac, H4K20me3, H3K56Ac, histone H4, p300, Tip60, HBO1, HAT1, CBP (Abcam), E‐cadherin, histone H3 (Cell Signaling Technology), plakoglobin, H3K4me2, H3K9Ac, H4K16Ac (Millipore), vimentin (Sigma‐Aldrich), and β‐actin (Genetex).
H3k56ac
Manufactured by Abcam
Sourced in United States
H3K56ac is a histone H3 acetylation mark that occurs at lysine 56 of the histone H3 protein. It is involved in various cellular processes, including DNA repair and chromatin structure regulation.
Automatically generated - may contain errors
Lab products found in correlation
H3k9ac, by Merck Group (4 mentions)
H3k18ac, by Abcam (2 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Vimentin, by Merck Group (1 mentions)
H3k4me2, by Merck Group (1 mentions)
Hif 1α, by BD (1 mentions)
N cadherin, by BD (1 mentions)
Plakoglobin, by Merck Group (1 mentions)
H4k16ac, by Merck Group (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Histone h4, by Abcam (1 mentions)
H4k20me3, by Abcam (1 mentions)
H4k12ac, by Abcam (1 mentions)
Tip60, by Abcam (1 mentions)
H4k8ac, by Abcam (1 mentions)
β actin, by GeneTex (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
Anti ha, by Roche (1 mentions)
Myc epitope, by Santa Cruz Biotechnology (1 mentions)
Recombinant histone h3, by Roche (1 mentions)
7 protocols using h3k56ac
1
RNA Pulldown and Histone Analysis
2
Purification and Analysis of Rtt109 Modifications
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His-tagged fusion proteins were prepared using Ni-NTA agarose beads (Qiagen, UK) according to the manufacturer’s protocol and HA epitope-tagged Pkc1p were prepared essentially as described previously (Darieva et al., 2012 (link)). Recombinant histone H3 was made commercially (Active Motif).
Pulldown assays with immunoprecipitated HA-Pkc1 from DZ2 cells and in vitro translated Rtt109 were carried out as described previously (Pic-Taylor et al., 2004 (link)).
To detect epitope-tagged derivatives by western analysis, anti-HA (Roche) and anti-tubulin TAT-1 (CRUK), H3K56ac (Active motif), H3K9ac (Abcam), H3K18ac (Abcam), H3 (Active motif), myc epitope (Santa Cruz), H3T45-P (Active motif) antibodies and Supersignal west dura substrate (Pierce) were used.
Rabbit polyclonal monospecific antibody against the phosphothreonine at amino acid 46 on yeast Rtt109 (Rtt109 T46-P) was generated from immunizing rabbits with a KLH-conjugated peptide (H-DDKRVPKST(PO3H2)IKTC-NH2), and then cross-affinity purification of polyclonal antibodies specific to modified Rtt109 T46-P or non-modified pRtt109 was performed (Eurogentec). For immunoblotting analysis we used 1:1000 dilution for both antibodies.
Pulldown assays with immunoprecipitated HA-Pkc1 from DZ2 cells and in vitro translated Rtt109 were carried out as described previously (Pic-Taylor et al., 2004 (link)).
To detect epitope-tagged derivatives by western analysis, anti-HA (Roche) and anti-tubulin TAT-1 (CRUK), H3K56ac (Active motif), H3K9ac (Abcam), H3K18ac (Abcam), H3 (Active motif), myc epitope (Santa Cruz), H3T45-P (Active motif) antibodies and Supersignal west dura substrate (Pierce) were used.
Rabbit polyclonal monospecific antibody against the phosphothreonine at amino acid 46 on yeast Rtt109 (Rtt109 T46-P) was generated from immunizing rabbits with a KLH-conjugated peptide (H-DDKRVPKST(PO3H2)IKTC-NH2), and then cross-affinity purification of polyclonal antibodies specific to modified Rtt109 T46-P or non-modified pRtt109 was performed (Eurogentec). For immunoblotting analysis we used 1:1000 dilution for both antibodies.
3
Histone Purification and Antibody Analysis
4
Immunoblot analysis of autophagy and epigenetic markers
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After treatments, cells were collected and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 250 mM NaCl, and 0.1% Triton, and completed with protease (ThermoScientific, A32953) and phosphatase inhibitors (ThermoScientific, 88667). Total protein extracts were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose filter, and subjected to immunoblot assay. Following primary antibodies were used: H3K9Ac (Millipore, 07–352); H3K56Ac (Abcam, ab76307); SIRT6 (Novus Biologicals, NBP1-30101); LC3B (Sigma-Aldrich, L7543); total AMP-activated protein kinase (AMPK; Cell Signaling, 2532) and phospho AMPK (Thr172; Cell Signaling, 2535); total (Calbiochem, ST1521) and phospho ULK1 (Ser555 and Ser757; Cell Signaling, 5869 and 6888 respectively); total mTOR (Cell Signaling, 4517) and phospho mTOR (ser2448; Cell Signaling, 2971); Beclin-1 (Cell Signaling, 3738); ATG5 (Cell Signaling, 2630); PARP1 (BD Pharmingen, 551025); β-actin (Sigma, A5441); HSP72/73 (Calbiochem, 386032); and H3 (Abcam, ab1791). Following secondary antibodies were used: Goat anti-mouse or anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase conjugated antibodies (Biorad, 1706516 and 1706515 respectively). Densitometry was performed using ImageJ software.
5
Quantification of DNA Damage Markers
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The antibodies used were 53BP1 (sc-22760, Santa Cruz Biotechnology, CA, USA), H4ac (05–1355, Millipore, Temecula, CA, USA), H3K56ac (ab76307, Abcam, Cambridge, UK), β-actin (sc-1616, Santa Cruz), and γH2AX (05-636-1; Millipore). Alexa Fluor® 546 (excitation/emission: 556 nm/570 nm) and Alexa Fluor® A488 (excitation/emission: 488 nm/519 nm) (A11010, Invitrogen Life Sciences, Carlsbad, CA, USA) were also used.
6
Histone Extraction and Detection Protocol
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Dissected brains were immediately frozen in liquid nitrogen and stored at −80°C. Frozen brains were manually homogenized in lysis buffer (10 mM Tris-HCl [pH 6.5], 50 mM sodium disulfite, 10 mM MgCl2, 10 mM sodium butyrate, 8.6% sucrose, 1% Triton X-100) supplemented with protease inhibitor cocktail (Roche) and PMSF. Histone isolation was performed as previously described (23 (link)). Equal amounts of histones (2 μg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Protran; Whatman) according to standard protocols. The following antibodies were used: H3 C-terminal (catalog number ab1791; Abcam), H3K14ac (catalog number 07-353; Millipore), H3K27ac (catalog number ab4729; Abcam), H3K4ac (catalog number 39381; Active Motif), H3K56ac (catalog number ab76307; Abcam), H3K9ac (catalog number 06-942; Millipore), H4K12ac (Sat44; Seiser laboratory), H4K16ac (Sat53; Seiser laboratory), and H4K8ac (Sat198; Seiser laboratory) antibodies.
7
Comprehensive Immunostaining Protocol for Glial Markers
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Immunostaining was performed with the following primary antibodies: H2A.Z.1 (Active-Motif, # 39943, Rabbit, 1:2000), GFAP (Sigma, G6171, Mouse, 1:10 000; Dako, Z033429, Rabbit, 1:3000), Acsbg1 (Abcam, ab 118154, Mouse, 1:1000), Aldh1l1 (Abcam, ab56777, Rabbit, 1:500), GLAST (Proteintech, 20785-1-AP, Rabbit, 1:1000), BLBP (Proteintech, 14836-1-AP, Rabbit, 1:000), S100β (Proteintech, 15146-1-AP, Rabbit, 1:500), Pax6 (Abcam, ab5790, Rabbit, 1:000), Sox2 (R&D,MAB2018, Mouse, 1:1000), BrdU (Abcam, ab6326, Rat, 1:1000), TUJ1 (Millipore, MAB1637, Rabbit, 1:2000), STAT3 (Cell Signaling Technology, 4904P, Mouse, 1:2000), phospho-STAT3 (Tyr705) (Cell Signaling Technology, 9145S, Rabbit, 1:500), β-Actin (Proteintech, 20536-1-AP, Rabbit, 1:5000), Flag (Sigma, F7425, Mouse, 1:2000), H2A (Proteintech, 10445-1-AP, Rabbit, 1:000), H3 (Cell Signaling Technology, 4499, Rabbit, 1:2000), H3K56ac (Abcam, ab76307, Rabbit, 1:2000), H3K9ac (Millipore, 07-352, Rabbit, 1:2000), H3K27ac (Millipore, 07-360, Rabbit, 1:2000), H3K4me3 (Millipore, 07-473, Rabbit, 1:2000), Tri-Methyl-Histone H3 (Lys36) (Cell Signaling Technology, # 9763, Rabbit, 1:2000), FOLR1 (Bioworld Technology, BS3861, Rabbit, 1:500).
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