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1.
Methods Mol Biol ; 567: 27-43, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19588083

RESUMO

We present here the very robust characterization and quality control (QC) process that we have established for our polyclonal antibodies, which are mainly directed against targets relevant to the epigenetics field such as modified histones, modifying enzymes, and chromatin-interacting proteins. The final purpose of the characterization and QC is to label antibodies as chromatin immunoprecipitation (ChIP) grade. Indeed, the ChIP method is extensively used in epigenetics to study gene regulation and relies on the use of antibodies to select the protein of interest and then precipitate and identify the DNA associated to it. We have optimized in-house all protocols and reagents needed from the first to the last step of antibody characterization. First, following immunizations, the rabbit crude serum is tested for immune response. Whether or not the antibody is specific is determined in further characterizations. Then, only specific antibodies are tested in ChIP using an optimized method which is ideal for antibody screening. Once QC is established for one antibody, it is used to similarly characterize each antibody batch in order to supply researchers in a reproducible manner with validated antibodies. All in all, this demonstrates that we develop epigenetics research tools based on everyday's researcher's needs by providing batch-specific fully characterized ChIP-grade antibodies.


Assuntos
Anticorpos/farmacologia , Imunoprecipitação da Cromatina/métodos , Imunoprecipitação da Cromatina/normas , Animais , Western Blotting/métodos , Imunoprecipitação da Cromatina/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Controle de Qualidade , Coelhos
2.
Methods Mol Biol ; 567: 237-47, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19588096

RESUMO

Epigenetics is the study of heritable changes in gene expression. Chromatin immunoprecipitation (ChIP) and methylation status analysis of genes have been applied to the study of epigenetic modifications, often perturbed in human cancer. ChIP is a technique allowing the analysis of the protein association with specific genomic regions in the context of intact cells. ChIP and immunoprecipitation (IP) of methylated DNA, both rely on the use of well-characterized specific antibodies. The first is described in Chapter 2 and the second is shown here. At Diagenode, a novel METHYL kit has been designed to immunoprecipitate methylated DNA (Methyl DNA IP). This kit allows you to perform DNA methylation analysis of your sample together with optimized internal IP controls, all in one tube. This brand new Methyl DNA IP method provides methylated DNA (meDNA) and unmethylated DNA (unDNA) controls to be used together with your DNA sample, allowing direct correlation between immunoprecipitated material and methylation status. Such methylation analysis is highly specific and each IP is quality controlled, two essential keys for reliable results. In addition, the kit protocol is fast and user-friendly.


Assuntos
Imunoprecipitação da Cromatina/métodos , Metilação de DNA/fisiologia , DNA/metabolismo , Animais , DNA/análise , Epigênese Genética/fisiologia , Humanos
3.
Mol Biol Cell ; 14(2): 445-59, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12589046

RESUMO

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2(AA)). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2(AA) mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2(AA). Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport.


Assuntos
Actinas/química , Diacetil/análogos & derivados , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Miosinas/química , Transporte Proteico , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Actinas/metabolismo , Animais , Transporte Biológico , Brefeldina A/farmacologia , Cálcio/metabolismo , Linhagem Celular , DNA Complementar/metabolismo , Diacetil/farmacologia , Inibidores Enzimáticos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/metabolismo , Miosinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Proteínas Recombinantes/metabolismo , Toxina Shiga/farmacologia , Fatores de Tempo , Proteínas do Envelope Viral/metabolismo , Quinases Associadas a rho
4.
Mol Biol Cell ; 14(2): 670-84, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12589062

RESUMO

Cell motility and cell polarity are essential for morphogenesis, immune system function, and tissue repair. Many animal cells move by crawling, and one main driving force for movement is derived from the coordinated assembly and disassembly of actin filaments. As tissue culture cells migrate to close a scratch wound, this directional extension is accompanied by Golgi apparatus reorientation, to face the leading wound edge, giving the motile cell inherent polarity aligned relative to the wound edge and to the direction of cell migration. Cellular proteins essential for actin polymerization downstream of Rho family GTPases include the Arp2/3 complex as an actin nucleator and members of the Wiskott-Aldrich Syndrome protein (WASP) family as activators of the Arp2/3 complex. We therefore analyzed the involvement of the Arp2/3 complex and WASP-family proteins in in vitro wound healing assays using NIH 3T3 fibroblasts and astrocytes. In NIH 3T3 cells, we found that actin and Arp2/3 complex contributed to cell polarity establishment. Moreover, overexpression of N-terminal fragments of Scar2 (but not N-WASP or Scar1 or Scar3) interfere with NIH 3T3 Golgi polarization but not with cell migration. In contrast, actin, Arp2/3, and WASP-family proteins did not appear to be involved in Golgi polarization in astrocytes. Our results thus indicate that the requirement for Golgi polarity establishment is cell-type specific. Furthermore, in NIH 3T3 cells, Scar2 and the Arp2/3 complex appear to be involved in the establishment and maintenance of Golgi polarity during directed migration.


Assuntos
Proteínas do Citoesqueleto/fisiologia , Complexo de Golgi/metabolismo , Proteínas dos Microfilamentos/fisiologia , Cicatrização , Células 3T3 , Proteína 2 Relacionada a Actina , Animais , Astrócitos/metabolismo , Western Blotting , Movimento Celular , DNA/metabolismo , Deleção de Genes , Ligantes , Camundongos , Proteínas dos Microfilamentos/química , Microscopia de Fluorescência , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Tempo , Família de Proteínas da Síndrome de Wiskott-Aldrich
5.
J Cell Sci ; 116(Pt 4): 743-56, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12538774

RESUMO

Scratch-wound assays are commonly used to study the ability of cells to polarize and migrate. In a previous study we showed that Golgi reorientation in response to a scratch wound is actin-dependent in NIH 3T3 cells but not in astrocytes. In this investigation, to study cell polarity and motility further, we used the polarization of the Golgi and microtubule organizing center (MTOC), as well as the ability of NIH 3T3 cells to migrate, in a scratch-wound assay. Unlike Golgi polarization, MTOC polarization was not dependent on actin, the Arp2/3 complex or Wiskott-Aldrich syndrome protein (WASP)-family proteins. By contrast, disruption of microtubules inhibited MTOC polarity, but not Golgi polarity. Migration was found to be dependent both on actin and microtubules. Expression of the formin-homology 2 (FH2) region of mDia1 inhibited Golgi polarization and migration but not MTOC polarization. Similarly, ST638, a Src inhibitor, inhibited Golgi polarization and migration but not MTOC polarization, whereas expression of the actin regulator IRSp53 only inhibited cell migration. Interestingly, the inhibition of cell migration by the mDia1 FH2 domain could be overcome by addition of Y27632, an inhibitor of ROCK (Rho-associated kinase). In fact, in the presence of ROCK inhibitor, cell migration was accelerated but polarization of both the Golgi and MTOC were inhibited. These data show that, in NIH 3T3 cells, different aspects of cell polarization and migration occur by different mechanisms, and both actin and microtubule networks are required. In addition, this study indicates that MTOC and Golgi polarization events are separately controlled.


Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Células Eucarióticas/metabolismo , Complexo de Golgi/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Células 3T3 , Actinas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Inibidores Enzimáticos/farmacologia , Células Eucarióticas/ultraestrutura , Forminas , Complexo de Golgi/ultraestrutura , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Centro Organizador dos Microtúbulos/ultraestrutura , Microtúbulos/ultraestrutura , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína/fisiologia , Cicatrização/fisiologia , Quinases Associadas a rho
7.
J Bacteriol ; 184(13): 3433-41, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12057936

RESUMO

Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The Shigella TTSS is encoded by the mxi/spa loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a Shigella spa32 mutant and studied its phenotype. The spa32 gene shows low sequence homology to Salmonella TTSS1 invJ/spaN and to flagellar fliK. The spa32 mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the spa32 mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the spa32 mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the spa32 mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the spa32 mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.


Assuntos
Adesinas Bacterianas , Proteínas de Bactérias/metabolismo , Proteínas de Membrana , Shigella flexneri/metabolismo , Proteínas de Bactérias/genética , Corantes/metabolismo , Vermelho Congo/metabolismo , Células HeLa/microbiologia , Humanos , Microscopia Eletrônica , Mutação , Shigella flexneri/genética , Shigella flexneri/patogenicidade , Especificidade por Substrato
8.
Cell Motil Cytoskeleton ; 51(4): 187-96, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11977093

RESUMO

Shigella move through the cytoplasm of host cells by active polymerization of host actin to form an "actin tail." Actin tail assembly is mediated by the Shigella protein IcsA. The process of Shigella actin assembly has been studied extensively using IcsA-expressing Escherichia coli in cytoplasmic extracts of Xenopus eggs. However, for reasons that have been unclear, wild type Shigella does not assemble actin in these extracts. We show that the defect in actin assembly in Xenopus extracts by Shigella can be rescued by increasing IcsA expression by approximately 3-fold. We calculate that the number of IcsA molecules required on an individual bacterium to assemble actin filaments in extracts is approximately 1,500-2,100 molecules, and the number of IcsA molecules required to assemble an actin tail is approximately 4,000 molecules. The majority of wild type Shigella do not express these levels of IcsA when grown in vitro. However, in infected host cells, IcsA expression is increased 3.2-fold, such that the number of IcsA molecules on a significant percentage of intracellular wild type Shigella would exceed that required for actin assembly in extracts. Thus, the number of IcsA molecules estimated from our studies in extracts as being required on an individual bacterium to assemble actin filaments or an actin tail is a reasonable prediction of the numbers required for these functions in Shigella-infected cells.


Assuntos
Actinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Shigella/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Biopolímeros/metabolismo , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Óvulo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Xenopus
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