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1.
Cancer Sci ; 111(1): 148-159, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733123

RESUMO

The majority of breast cancers are primarily hormone-sensitive and can be managed by endocrine therapy, although therapy-resistant or hormone-refractory cancers need alternative treatments. Recently, increasing attention is being paid to RNA-binding proteins (RBP) in cancer pathophysiology. The precise role of RBP in breast cancer, however, remains to be clarified. We herein show that an RBP non-POU domain-containing octamer binding (NONO) plays a critical role in the pathophysiology of breast cancers regardless of their hormone dependency. Clinicopathological and immunohistochemical study of 127 breast cancer cases showed that NONO is a significant independent prognostic factor for breast cancer patients. Notably, siRNA-mediated NONO knockdown substantially repressed the proliferation of both hormone-sensitive MCF-7 and hormone-refractory MB-MDA-231 breast cancer cells. Integrative analysis combined with expression microarray and RIP-sequencing (RNA immunoprecipitation-sequencing) showed that NONO post-transcriptionally regulates the expression of cell proliferation-related genes by binding to their mRNAs, as exemplified by S-phase-associated kinase 2 and E2F transcription factor 8. Overall, these results suggest that NONO is a key regulator for breast cancer proliferation through the pre-mRNA splicing of cell proliferation-related genes and could be a potential new diagnostic and therapeutic target for advanced disease.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Proteínas Quinases Associadas a Fase S/genética , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica/genética , Humanos , Imunoprecipitação/métodos , Células MCF-7 , RNA Mensageiro/genética
2.
Mol Immunol ; 116: 63-72, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31622795

RESUMO

Somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID) and requires target gene transcription. A splice isoform of SRSF1, SRSF1-3, is necessary for AID-dependent SHM of IgV genes. Nevertheless, its exact molecular mechanism of action in SHM remains unknown. Our in silico studies show that, unlike SRSF1, SRSF1-3 lacks a strong nuclear localization domain. We show that the absence of RS domain in SRSF1-3 affects its nuclear localization, as compared to SRSF1. Consequently, SRSF1-3 is predominantly present in the cytoplasm. Remarkably, co-immunoprecipitation studies showed that SRSF1-3 interacts with Topoisomerase 1 (TOP1), a crucial regulator of SHM that assists in generating ssDNA for AID activity. Moreover, the immunofluorescence studies confirmed that SRSF1-3 and TOP1 are co-localized in the nucleus. Furthermore, Proximity Ligation Assay corroborated the direct interaction between SRSF1-3 and TOP1. An interaction between SRSF1-3 and TOP1 suggests that SRSF1-3 likely influences the TOP1 activity and consequently can aid in SHM. Accordingly, SRSF1-3 probably acts as a link between TOP1 and SHM, by spatially regulating TOP1 activity at the Ig locus. We also confirmed the interaction between SRSF1-3 and AID in chicken B-cells. Thus, SRSF1-3 shows dual-regulation of SHM, via interacting with AID as well as TOP1.


Assuntos
Citidina Desaminase/genética , DNA Topoisomerases Tipo I/genética , Genes de Imunoglobulinas/genética , Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Hipermutação Somática de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Linhagem Celular , Núcleo Celular/genética , Galinhas/genética , Switching de Imunoglobulina , Imunoprecipitação/métodos , Camundongos , Isoformas de Proteínas/genética
3.
PLoS Biol ; 17(7): e3000373, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31329577

RESUMO

Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isoforms have functionally specialized to bind distinct proteins remains unclear. Here, we describe a comprehensive protein-protein interaction resource, obtained using in planta immunoprecipitation (IP) followed by mass spectrometry (MS), to define the potato ATG8 interactome. We discovered that ATG8 isoforms bind distinct sets of plant proteins with varying degrees of overlap. This prompted us to define the biochemical basis of ATG8 specialization by comparing two potato ATG8 isoforms using both in vivo protein interaction assays and in vitro quantitative binding affinity analyses. These experiments revealed that the N-terminal ß-strand-and, in particular, a single amino acid polymorphism-underpins binding specificity to the substrate PexRD54 by shaping the hydrophobic pocket that accommodates this protein's ATG8-interacting motif (AIM). Additional proteomics experiments indicated that the N-terminal ß-strand shapes the broader ATG8 interactor profiles, defining interaction specificity with about 80 plant proteins. Our findings are consistent with the view that ATG8 isoforms comprise a layer of specificity in the regulation of selective autophagy pathways in plants.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Imunoprecipitação/métodos , Espectrometria de Massas/métodos , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas/classificação , Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Conformação Proteica em Folha beta , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteômica/métodos , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Tabaco/genética , Tabaco/metabolismo
4.
Lab Chip ; 19(11): 1922-1928, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31073561

RESUMO

Heterogeneity in a tumor allows a small portion of cancer cells to survive and regrow upon targeted cancer therapy, eventually leading to cancer relapse. Such drug-resistant cells often exhibit dynamic adaptation of their signaling pathways at the level of protein-protein interactions (PPIs). To probe the rewiring of signaling pathways and the heterogeneity across individual cancer cells, we developed a single-cell version of the co-immunoprecipitation (co-IP) analysis that examines the amount and PPIs of target proteins immunoprecipitated from individual cells. The method captures cancer cells at predefined locations using a microfluidic chip, pulls down target proteins on the surface using antibodies, and lyses the captured cells in situ. Then, subsequent addition of eGFP-labeled downstream proteins enables the determination of the corresponding PPIs for the minimal amount of target proteins sampled from a single cell. We applied the technique to probe epidermal growth factor receptors (EGFRs) in PC9 lung adenocarcinoma cells. The results reveal that the strength of EGFR PPIs can be largely uncorrelated with the expression level of EGFRs in single cells. In addition, the individual PC9 cells showed markedly different patterns of PPIs, indicating a high heterogeneity in EGFR signaling within a genetically homogeneous population.


Assuntos
Imunoprecipitação/métodos , Mapeamento de Interação de Proteínas/métodos , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Fenótipo , Transdução de Sinais
5.
Nat Protoc ; 14(6): 1734-1755, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31053798

RESUMO

R-loops are prevalent three-stranded non-B DNA structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops are implicated in various basic nuclear processes, such as class-switch recombination, transcription termination and chromatin patterning. Perturbations in R-loop metabolism have been linked to genomic instability and have been implicated in human disorders, including cancer. As a consequence, the accurate mapping of these structures has been of increasing interest in recent years. Here, we describe two related immunoprecipitation-based methods for mapping R-loop structures: basic DRIP-seq (DNA-RNA immunoprecipitation followed by high-throughput DNA sequencing), an easy, robust, but resolution-limited technique; and DRIPc-seq (DNA-RNA immunoprecipitation followed by cDNA conversion coupled to high-throughput sequencing), a high-resolution and strand-specific iteration of the method that permits accurate R-loop mapping genome wide. Briefly, after gentle DNA extraction and restriction digestion with a cocktail of enzymes, R-loop structures are immunoprecipitated with the anti-RNA-DNA hybrid S9.6 antibody. Compared with DRIP-seq, in which the immunoprecipitated DNA is directly sequenced, DRIPc-seq permits the recovery of the RNA moiety of R-loops, and these RNA strands are subjected to strand-specific RNA sequencing (RNA-seq) analysis. DRIPc-seq can be performed in 5 d and can be applied to any cell type, provided sufficient starting material can be collected. Accurately mapping R-loop distribution in various cell lines and under varied conditions is essential to understanding the formation, roles and dynamic resolution of these important structures.


Assuntos
DNA/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , RNA/análise , Animais , Anticorpos/química , Anticorpos Monoclonais/química , DNA/genética , Humanos , Camundongos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , RNA/genética
6.
Anal Bioanal Chem ; 411(14): 3009-3019, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31076819

RESUMO

The N-glycosylation of proteins is one of the most important post-translational modifications relevant to various biological functions. The identification and quantification of N-glycoproteins in liquid chromatography-mass spectrometry (LC-MS) is challenging because of their low analytical sensitivity and selectivity. This is due to their microheterogeneity and the difficulty of synthesizing N-glycopeptides as an internal standard. Parallel reaction monitoring (PRM) is widely used in targeted LC-MS. The key advantage of LC-PRM is that it can identify N-glycopeptides using tandem mass spectrometry (MS/MS) fragmentation, even without an internal standard. We investigated the feasibility of analyzing N-glycoproteins using multiplex immunoprecipitation to improve sensitivity and selectivity. We targeted N-glycoproteins [α-fetoprotein (AFP), vitronectin (VTN), and α-1-antichymotrypsin (AACT)] that are abnormally glycosylated in hepatocellular carcinoma (HCC). Their tryptic N-glycopeptides were selected to determine the percentages of fucosylated N-glycopeptides using Y ions, which include glycopeptide fragments with amino acid sequences. Finally, we confirmed that the area under the receiver operating characteristic curve (AUC = 0.944) for the combination of AFP and VTN increased more so than for a single glycopeptide (AUC = 0.889 for AFP and 0.792 for VTN) with respect to discriminating between HCC and cirrhosis serum. This study shows that an LC-PRM method using multiplex N-glycoproteins immunoprecipitated from serum could be applied to develop and verify cancer biomarkers. Graphical abstract.


Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Hepatocelular/diagnóstico , Cromatografia Líquida/métodos , Glicoproteínas/sangue , Imunoprecipitação/métodos , Neoplasias Hepáticas/diagnóstico , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Calibragem , Carcinoma Hepatocelular/sangue , Estudos de Casos e Controles , Estudos de Viabilidade , Fucose/química , Glicoproteínas/química , Glicoproteínas/normas , Glicosilação , Humanos , Limite de Detecção , Neoplasias Hepáticas/sangue , Curva ROC , Padrões de Referência , Vitronectina/sangue , alfa 1-Antiquimotripsina/sangue , alfa-Fetoproteínas/metabolismo
7.
Biomed Res Int ; 2019: 1856180, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019965

RESUMO

Objective: This study aimed to clarify the clinical features, the serum level of autoantibodies, and cytokine of myositis patients with anti-EJ antibody, which targets glycyl tRNA-synthetase (GlyRS). Methods: Sera of 236 Chinese patients with myositis were screened for anti-EJ by a novel immunoprecipitation assay of flag-tagged GlyRS. Anti-EJ positive patients are evaluated for the clinical features and cytokine profile. Results: The sera from 4 of 236 adult myositis patients were found to carry the anti-EJ using established novel immunoprecipitation assay and immunoblotting. The prevalence of anti-EJ in our cohorts is about 1.7%. The decline of anti-EJ level was detected in two patients during disease remission. Interstitial lung disease and muscle weakness, but not skin involvement, are common clinical features of anti-EJ positive patients. Moreover, using a cytokine profile analyses, we found that the serum levels of IP-10, IL-6, MCP-1, and VEGF were significantly elevated in patients with anti-EJ and gradually decreased during disease remission of two patients, whereas IL-8 level was obviously reduced in these patients. Conclusion: The novel immunoprecipitation assay is suitable to detect and monitor the levels of anti-EJ autoantibody. The serum levels of anti-EJ, IP-10, IL-6, MCP-1, and VEGF may be related to disease activity in myositis patients with anti-EJ antibodies.


Assuntos
Autoanticorpos/sangue , Citocinas/sangue , Miosite/sangue , Adulto , Autoanticorpos/imunologia , Citocinas/imunologia , Feminino , Humanos , Imunoprecipitação/métodos , Doenças Pulmonares Intersticiais/sangue , Doenças Pulmonares Intersticiais/imunologia , Masculino , Pessoa de Meia-Idade , Miosite/imunologia
8.
Methods Mol Biol ; 1933: 279-288, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945192

RESUMO

Recent advances in next-generation sequencing have revealed that majority of the plant genome is transcribed into long noncoding RNA (lncRNA). Many lncRNAs function by interacting with proteins and forming regulatory complexes. RNA-protein interactions are vital in controlling core cellular processes like transcription and translation. Therefore, identifying proteins that interact with lncRNAs is the first step to deciphering lncRNA functions. Here, we describe an RNA-protein pull-down assay, which enables the identification of proteins that interact with an RNA under study. As an example, we describe pull-down of proteins interacting with lncRNA ELENA1, which promotes the enrichment of MED19a on PR1 promoter to activate PR1 expression.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Imunoprecipitação/métodos , Extratos Vegetais/metabolismo , RNA Longo não Codificante/metabolismo , RNA de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Extratos Vegetais/genética , RNA Longo não Codificante/genética , RNA de Plantas/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma
9.
Methods Mol Biol ; 1933: 289-295, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945193

RESUMO

Long noncoding RNAs (lncRNAs) play important roles in several processes including control of gene expression. These RNAs function through binding to histone-modifying complexes and transcriptional machinery including transcription factor, mediator, and RNA polymerase II. We present methods for the discovery and characterization of lncRNAs. RNA immunoprecipitation (RIP) is a modified version of chromatin immunoprecipitation (ChIP), and it is now generally used in lncRNA study. The method allows for testing of lncRNA-protein interactions in vivo. RIP assay facilitates the identification of consensus sequences of preferred binding site for the RNA-binding protein under study, and identification of the binding sites can provide valuable information on the possible mechanism by which the RNA-binding protein functions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Imunoprecipitação da Cromatina/métodos , Imunoprecipitação/métodos , RNA Longo não Codificante/metabolismo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Longo não Codificante/genética , RNA de Plantas/genética , Proteínas de Ligação a RNA/genética
10.
Methods Mol Biol ; 1933: 389-394, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945199

RESUMO

Transcriptome-wide mapping RNA modification is crucial to understand the distribution and function of RNA modifications. Here, we describe a protocol to transcriptome-wide mapping 5-methylcytosine (m5C) in plant, by a RNA immunoprecipitation followed by deep sequencing (m5C-RIP-seq) approach. The procedure includes RNA extraction, fragmentation, RNA immunoprecipitation, and library construction.


Assuntos
5-Metilcitosina/química , Arabidopsis/genética , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , RNA de Plantas/genética , Transcriptoma , Biologia Computacional , RNA de Plantas/química , Análise de Sequência de RNA/métodos
11.
PLoS One ; 14(4): e0215340, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30970003

RESUMO

Estrogen Receptor alpha (ERα) plays a major role in most breast cancers, and it is the target of endocrine therapies used in the clinic as standard of care for women with breast cancer expressing this receptor. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used against the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.


Assuntos
Anticorpos , Receptor alfa de Estrogênio/imunologia , Imunoprecipitação/métodos , Especificidade de Anticorpos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Células MCF-7
12.
Int J Mol Med ; 43(6): 2267-2278, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31017262

RESUMO

Among a number of mRNA modifications, N6­methyladenosine (m6A) modification is the most common type in eukaryotes and nuclear­replicating viruses. m6A has a significant role in numerous cancer types, including leukemia, brain tumors, liver cancer, breast cancer and lung cancer. Although m6A methyltransferases are essential during RNA modifications, the biological functions of m6A and the underlying mechanisms remain to be fully elucidated, predominantly due to the limited detection methods for m6A. In the present review, the currently available m6A detection methods and the respective scope of their applications are presented to facilitate the further investigation of the roles of m6A in biological process.


Assuntos
Adenosina/análogos & derivados , RNA/química , Adenosina/análise , Adenosina/genética , Animais , Técnicas Biossensoriais/métodos , Northern Blotting/métodos , Cromatografia Líquida de Alta Pressão/métodos , Técnicas Eletroquímicas/métodos , Humanos , Immunoblotting/métodos , Imunoprecipitação/métodos , Metilação , Neoplasias/genética , RNA/genética , Análise de Sequência de RNA/métodos
13.
Methods Mol Biol ; 1969: 33-49, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30877668

RESUMO

Deep sequencing technology has revolutionized transcriptome analyses of both prokaryotes and eukaryotes. RNA-sequencing (RNA-seq), which is based on massively parallel sequencing of cDNAs, has been used to annotate transcript boundaries and has revealed widespread antisense transcription as well as a wealth of novel noncoding transcripts in many bacterial pathogens. Moreover, RNA-seq is nowadays also widely used to comprehensively explore the interaction between RNA-binding proteins and their RNA targets on a genome-wide level in many human-pathogenic bacteria. In particular, immunoprecipitation of an RNA-binding protein (RBP) of interest followed by isolation and analysis of all bound RNAs (RNA immunoprecipitation (RIP)) allows rapid characterization of its RNA regulon. Here, we describe an experimental approach which employs co-immunoprecipitation (coIP) of the RNA-binding chaperone Hfq along with bound RNAs followed by deep-sequencing of co-purified RNAs (RIP-Seq) from a genetically modified strain of Neisseria meningitidis expressing a chromosomally encoded Hfq-3×FLAG protein. This approach allowed us to comprehensively identify both mRNAs and sRNAs as targets of Hfq and served as an excellent starting point for sRNA research in this human pathogenic bacterium.


Assuntos
Perfilação da Expressão Gênica/métodos , Fator Proteico 1 do Hospedeiro/metabolismo , Imunoprecipitação/métodos , Neisseria meningitidis/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Humanos , Neisseria meningitidis/genética , Neisseria meningitidis/isolamento & purificação , Ligação Proteica , RNA Bacteriano/genética , RNA Mensageiro/genética
14.
Prostate ; 79(6): 657-666, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30714180

RESUMO

BACKGROUND: DHX15 is a member of the DEAH-box (DHX) RNA helicase family. Our previous study identified it as an AR coactivator which contributes to prostate cancer progression. METHODS: We investigated DHX15 expression in castration resistant prostate cancer specimens and the influence of DHX15 on the responsiveness of prostate cancer cells to DHT stimulation. We also explored the role DHX15 played in enzalutamide resistance and the interacting domain in DHX15 with AR. DHX15 expression level in human CRPC specimens and prostate cancer specimens was detected by tissue microarray (TMA) immunostaining analysis. Colony formation assay was performed to determine the proliferation of cells treated with enzalutamide or DHT. siRNAs were used to knockdown DHX15. The interactions between DHX15 and AR were detected using co-immunoprecipitation assay. RESULTS: The expression level of DHX15 was upregulated in human CRPC specimens compared with hormone naïve prostate cancer specimens. DHX15 knockdown reduced AR sensitivity to low DHT concentrations in C4-2 cells. Inactivation of DHX15 sensitizes the enzalutamide treatment in C4-2 cells. Deletion mutagenesis indicated that DHX1 5 interacts with AR through its N terminal domain. CONCLUSIONS: These findings suggest that DHX15 contributes to prostate cancer progression. DHX15 is required for androgen receptor sensitivity to low DHT concentrations and contributes to enzalutamide resistance in C4-2 cells. Targeting DHX15 may improve the ADT treatment.


Assuntos
Feniltioidantoína/análogos & derivados , Neoplasias de Próstata Resistentes à Castração , RNA Helicases , Receptores Androgênicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoprecipitação/métodos , Masculino , Feniltioidantoína/farmacologia , Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , RNA Helicases/genética , RNA Helicases/metabolismo , Ativação Transcricional , Regulação para Cima
16.
Methods Mol Biol ; 1914: 131-143, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30729463

RESUMO

This chapter describes the analysis of signaling pathways in bone cells by the use of western blotting and immunoprecipitation, including a step-by-step guide to cell culture techniques, cellular and subcellular fractionation, protein isolation, purification, measurement, electrophoretic transfer, and detection.


Assuntos
Western Blotting/métodos , Osso e Ossos/citologia , Imunoprecipitação/métodos , Proteínas/análise , Transdução de Sinais , Animais , Western Blotting/instrumentação , Osso e Ossos/metabolismo , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Cultivadas , Eletroforese/instrumentação , Eletroforese/métodos , Humanos , Imunoprecipitação/instrumentação , Proteínas/isolamento & purificação , Proteínas/metabolismo
18.
Methods Cell Biol ; 149: 205-213, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30616821

RESUMO

ßarrestin1 and -2 (also known as arrestin2 and -3, respectively) are G protein-coupled receptor (GPCR) adapter proteins, performing three major functions in the cell: functional desensitization, i.e., G protein uncoupling from the receptor, GPCR internalization via clathrin-coated pits, and formation of signalosomes. The ßarrestins elicit a large part of the G protein-independent signaling emanating from GPCRs. Several methodologies have been developed over the past 15 years or so to quantify the GPCR-arrestin interaction/binding, especially since the latter's roles in signal transduction were discovered. One of the simplest and most traditional of these methodologies is the assay of co-immunoprecipitation (co-IP), followed by western blotting. This assay is also one of the most reliable ones, since it does not require any chemical modification of either component in the complex (i.e., neither of the receptor nor of the arrestin). Therefore, it is the only assay that can detect and semi-quantify interactions between native GPCRs and native arrestins. The caveat of this assay is of course that its reliability depends on the quality (specificity and sensitivity) of the utilized antibodies. Here, we describe a simple protocol for performing this co-IP assay to get a measurement of the steady-state levels of agonist-elicited GPCR-arrestin interaction in cells.


Assuntos
Arrestinas/metabolismo , Membrana Celular/metabolismo , Imunoprecipitação/métodos , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Humanos , Camundongos , Ligação Proteica , Ratos
19.
Methods Mol Biol ; 1925: 145-155, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30674024

RESUMO

Lysosomes are emerging as calcium store organelles that can modulate various intracellular processes such as the regulation of nutrient signaling through the activation of TFEB, a master gene for lysosomal function, or very specialized functions like lysosomal exocytosis. Here, we describe two different techniques that can be used to study these processes. In the case report, we described two studies where these methodologies allowed us to unravel the role of calcineurin in the dephosphorylation of TFEB as well as the involvement of TFEB in lysosomal exocytosis, respectively.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Calcineurina/metabolismo , Cálcio/metabolismo , Lisossomos/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Cátions Bivalentes/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular , Exocitose , Células HeLa , Humanos , Immunoblotting/métodos , Imunoprecipitação/métodos , Lisossomos/genética , Camundongos , Fosforilação , Transfecção/métodos
20.
Chemistry ; 25(14): 3455-3464, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30347476

RESUMO

N6 -Methyladenosine (m6 A) is the most abundant internal modification in eukaryotic mRNA. Specific m6 A reader and eraser proteins link this modification to many aspects of mRNA metabolism and regulate its levels in a dynamic way. Precise localization and quantification in varying biological samples is, therefore, relevant to understand the functional role of m6 A and mechanisms governing its regulation. In this Minireview, we summarize established and emerging concepts for m6 A mapping. Starting with the seminal m6 A-sequencing techniques based on immunoprecipitation, we will highlight technical improvements by photo-cross-linking and remaining challenges. As an alternative, antibody-free approaches will be presented. These include wild-type or engineered m6 A-sensitive enzymes and chemical biology approaches combining substrate analogues, chemical derivatization, and enzymatic steps to trace m6 A. Finally, single-molecule sequencing as a new avenue for direct detection of mRNA modifications will be discussed.


Assuntos
Adenosina/análogos & derivados , RNA Mensageiro/química , Adenosina/análise , Adenosina/genética , Adenosina/metabolismo , Animais , Anticorpos/química , Perfilação da Expressão Gênica/métodos , Humanos , Imunoprecipitação/métodos , Metilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma
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