Rapid and effective Western blotting of histones from acid‐urea‐Triton and sodium docecyl sulfate polyacrylamide gels: Two different approaches depending on the subsequent qualitative or quantitative analysis

Thiriet, Christophe; Albert, Philippe

doi:10.1002/elps.1150160161

Cited by 20 publications

(15 citation statements)

References 26 publications

(18 reference statements)

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“…The AUT strips were placed on sodium dodecyl sulfate (SDS)-15% polyacrylamide gels and blotted on nitrocellulose (0.45-m pore size), using Mini-Protean II electrophoresis and blot cells (Bio-Rad, La Jolla, Calif.). After equilibration of the gel for 30 min in 50 mM acetic acid and 0.5% (wt/vol) SDS, blotting was performed in 25 mM 3-(cyclohexylamino)-1-propanesulfonic acid buffer (pH 10) with 20% (vol/vol) methanol according to the protocol described by Thiriet and Albert (48). ubi-H2A was detected as described by Baarends et al (1).…”

Section: Methodsmentioning

confidence: 99%

Silencing of Unpaired Chromatin and Histone H2A Ubiquitination in Mammalian Meiosis

Baarends¹,

Wassenaar²,

Laan

et al. 2005

Molecular and Cellular Biology

285

261

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During meiotic prophase in male mammals, the X and Y chromosomes are incorporated in the XY body. This heterochromatic body is transcriptionally silenced and marked by increased ubiquitination of histone H2A. This led us to investigate the relationship between histone H2A ubiquitination and chromatin silencing in more detail. First, we found that ubiquitinated H2A also marks the silenced X chromosome of the Barr body in female somatic cells. Next, we studied a possible relationship between H2A ubiquitination, chromatin silencing, and unpaired chromatin in meiotic prophase. The mouse models used carry an unpaired autosomal region in male meiosis or unpaired X and Y chromosomes in female meiosis. We show that ubiquitinated histone H2A is associated with transcriptional silencing of large chromatin regions. This silencing in mammalian meiotic prophase cells concerns unpaired chromatin regions and resembles a phenomenon described for the fungus Neurospora crassa and named meiotic silencing by unpaired DNA.Chromatin remodeling is at the basis of control of cellspecific gene expression, cell determination, and differentiation. The nucleosome units of chromatin consist of two each of the histones H2A, H2B, H3, and H4. The N-terminal ends of these core histones extend from the nucleosome and can undergo posttranslational covalent modifications, such as methylation, acetylation, phosphorylation, and ADP-ribosylation of specific amino acid residues. Together, these modifications constitute the so-called histone code (45). Interaction of other nuclear proteins with chromatin is dependent on the histone code at specific chromatin regions and determines local chromatin structure, which can be open or closed. A remarkable component of the histone code is ubiquitination of C-terminal lysine residues of histones H2A and H2B. Ubiquitin, a protein of 7 kDa, can be attached to lysine residues of a specific protein substrate through the action of a multienzyme complex containing ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin ligase (E3) enzymes. Polyubiquitination can target proteins for degradation by the proteasome (34, 35). Monoubiquitination of histones, however, is a stable modification that does not decrease the half-life of the target histone (56).In the yeast Saccharomyces cerevisiae, histone H2A ubiquitination is not required for cell growth or sporulation (47), but histone H2B ubiquitination is an essential mechanism involved in sporulation (37). Most importantly, it has been shown that ubiquitination of H2B by the ubiquitin-conjugating enzyme RAD6, interacting with the ubiquitin ligase BRE1, is a prerequisite for dimethylation of histone H3 at lysine residues 4 and 79 (5,12,37,46). This mechanism is thought to be associated with potentiation of gene activation. It is not known whether this "trans-histone" mechanism is conserved between yeast and mammals. RAD6 shows marked evolutionary conservation. The two mammalian homologs of yeast RAD6, Hr6a/Ube2a and Hr6b/Ube2b, both show approximately 70% amino...

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Section: Methodsmentioning

confidence: 99%

Silencing of Unpaired Chromatin and Histone H2A Ubiquitination in Mammalian Meiosis

Baarends¹,

Wassenaar²,

Laan

et al. 2005

Molecular and Cellular Biology

285

261

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“…Extracted histones were then precipitated with 25% trichloroacetic acid (TCA), washed once with 99:1 acetone͞5 M HCl, and then once with acetone before resuspending in 8 M urea. Samples containing equal concentrations of histones were subjected to electrophoresis in 18% SDS͞ PAGE gels (30:0.8) acrylamide͞bisacrylamide, and then transferred to Immobilon-P (Millipore) in transfer buffer with 0.1% SDS (32). The blots were probed with rabbit polyclonal antisera recognizing H4 acetyl-lysine 5 (R40͞5), H4 acetyl-lysine 8 (R12͞ 8), H4 acetyl-lysine 12 (R20͞12), H4 acetyl-lysine 16 (R14͞16), H3 acetyl-lysine 9 and 18 (R48͞9,18), or H3 acetyl-lysine 14 (R224͞14).…”

Section: Methodsmentioning

confidence: 99%

HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription

Rundlett

Carmen

Kobayashi

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

591

552

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Increased histone acetylation has been correlated with increased transcription, and regions of heterochromatin are generally hypoacetylated. In investigating the cause-and-effect relationship between histone acetylation and gene activity, we have characterized two yeast histone deacetylase complexes. Histone deacetylase-A (HDA) is an Ϸ350-kDa complex that is highly sensitive to the deacetylase inhibitor trichostatin A. Histone deacetylase-B (HDB) is an Ϸ600-kDa complex that is much less sensitive to trichostatin A. The HDA1 protein (a subunit of the HDA activity) shares sequence similarity to RPD3, a factor required for optimal transcription of certain yeast genes. RPD3 is associated with the HDB activity. HDA1 also shares similarity to three new open reading frames in yeast, designated HOS1, HOS2, and HOS3. We find that both hda1 and rpd3 deletions increase acetylation levels in vivo at all sites examined in both core histones H3 and H4, with rpd3 deletions having a greater impact on histone H4 lysine positions 5 and 12. Surprisingly, both hda1 and rpd3 deletions increase repression at telomeric loci, which resemble heterochromatin with rpd3 having a greater effect. In addition, rpd3 deletions retard full induction of the PHO5 promoter fused to the reporter lacZ. These data demonstrate that histone acetylation state has a role in regulating both heterochromatic silencing and regulated gene expression.

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“…The proteins were transferred onto nitrocellulose membrane (8 Â 9 cm, 22 mm pore size, Osmonics Inc.) positioned at the anode side of the gel, with transfer buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS, pH 8.3 and 20% v/v methanol), in a Bio-Rad mini transblot apparatus at constant 100 V for 10 min followed by constant 60 V for 50 min. 55 After transfer, the nitrocellulose membrane was probed with a 1 : 1000 dilution of antiacetylHistone H3 antibody (Cat # 06-599; Upstate Biotechnology, Lake Placid, NY, USA), 1 : 1000 dilution of antihistone H3 antibody (Cat # 05-499; Upstate Biotechnology), 1 : 1000 dilution of antiacetyl-Histone H4 antibody (Cat # 06-598; Upstate Biotechnology) or 1 : 1000 dilution of antihistone H4 antibody (Cat # 07-108; Upstate Biotechnology), followed by a 1 : 5000 dilution of horseradish peroxidase-conjugated secondary antibody against respective rabbit IgG and mouse IgG and autoradiographed with Super Signal West Dura extended duration substrate (Cat # 34075; Pierce, Rockford, IL, USA). Blots were stripped with Western Re-Probe TM Buffer (Cat # 786-119; Geno Technology, St. Louis, MO, USA).…”

Section: Separation and Analysis Of Histonesmentioning

confidence: 99%

Histone deacetylase inhibitors differentially stabilize acetylated p53 and induce cell cycle arrest or apoptosis in prostate cancer cells

et al. 2005

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In LNCaP prostate cancer cells CG-1521, a new inhibitor of histone deacetylases, alters the acetylation of p53 in a sitespecific manner. While p53 is constitutively acetylated at Lys320 in LNCaP cells, treatment with CG-1521, stabilizes the acetylation of p53 at Lys373, elevating p21 (and inducing cell cycle arrest). Treatment with CG-1521 also promotes Bax translocation to the mitochondria and cleavage, and apoptosis. TSA stabilizes the acetylation of p53 at Lys382, elevating p21 levels and inducing cell cycle arrest, but does not induce Bax translocation or apoptosis. In LNCaP cells CG-1521, but not TSA, promotes the rapid degradation of HDAC2. These data suggest that the acetylation of p53 at Lys373 is required for the p53-mediated induction of cell cycle arrest and apoptosis, while acetylation of p53 at Lys382 induces only cell cycle arrest. In p53 À/À PC3 cells both compounds induce p21 and cell cycle arrest, but not Bax translocation or apoptosis, suggesting that both compounds can also induce p21 through a p53-independent mechanism.

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Rapid and effective Western blotting of histones from acid‐urea‐Triton and sodium docecyl sulfate polyacrylamide gels: Two different approaches depending on the subsequent qualitative or quantitative analysis

Cited by 20 publications

References 26 publications

Silencing of Unpaired Chromatin and Histone H2A Ubiquitination in Mammalian Meiosis

Silencing of Unpaired Chromatin and Histone H2A Ubiquitination in Mammalian Meiosis

HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription

Histone deacetylase inhibitors differentially stabilize acetylated p53 and induce cell cycle arrest or apoptosis in prostate cancer cells

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