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1.
Turkiye Parazitol Derg ; 43(Suppl 1): 13-17, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591873

RESUMO

Objective: Alveolar echinococcosis (AE) is one of the most lethal parasitic zoonoses in the Northern Hemisphere, and early serological detection is important to start treatment and to improve survival. A total of 50 sera samples of patients diagnosed as having various diseases were examined for by two different serological diagnostic methods. Methods: Em2-Em18 ELISA (Bordier Affinity Products, Crissier, Switzerland) and Echinococcus Western Blot immünoglobulin G (IgG) (LDBIO Diagnostics, Lyon, France) were used for analyisis. Results: A high titer of antibodies was found in 9 of 10 patients diagnosed as having AE with Em2-Em18 ELISA, in 2 of 21 patients with cystic echinococcosis, in 1 of 2 patients with fascioliasis and in 1 patient with chronic hepatitis. The Echinococcus Western Blot IgG test, used as a confirmatory test, showed IgG antibody in 85.7% (18/21) of patients with CE, while all serum samples of 10 patients with AE were evaluated as positive. This method yielded an incorrect diagnosis in the patient with chronic hepatitis and in the patient with granulomatous inflammation with caseification. Samples taken from patients with liver-related diseases and other parasitic-related diseases were found to be negative. Conclusion: The serological methods used in the study were found to be important in the early diagnosis of alveolar echinococcosis in the endemic areas, since it could be used in sero-epidemiological studies.


Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Equinococose/diagnóstico , Echinococcus/imunologia , Imunoglobulina G/sangue , Adulto , Animais , Western Blotting/métodos , Diagnóstico Diferencial , Equinococose Hepática/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Fasciolíase/diagnóstico , Feminino , Proteínas de Helminto/imunologia , Hepatite Crônica/diagnóstico , Humanos , Masculino
2.
Ann Clin Lab Sci ; 49(4): 507-512, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31471341

RESUMO

We report that the quantitative western blot (qWB) analysis requires a target protein-specific approach, and we provide a workflow that streamlines development of this process. First, the optimal primary antibody dilution is determined. Blots containing 15 µg total protein per lane are probed with the primary antibody at three concentrations and a secondary antibody concentration that is defined by the manufacturer. The lowest primary antibody concentration that detects a discrete band at the correct molecular weight is used in the remaining two steps. Secondly, the optimal protein load is determined. Blots containing 3.75 to 60 µg protein per lane are probed using the antibody concentrations defined in step 1. A target protein band intensity vs. protein load plot is used to determine the linear dynamic range (LDR) for the target protein. The midpoint of the LDR is defined as the optimal protein load. Finally, an appropriate loading control (LC) is identified. We found that the LDR for ß-actin, a commonly used LC, exhibited a narrow range, 3.75 to 15 µg. In contrast, the total protein assessed by a Ponceau staining method exhibited a broader LDR, 3.75 to 60 µg. Thus, the total protein is used as a LC. We conclude that the sensitivity and accuracy of the qWB method is dependent on the use of an optimal: 1) primary antibody dilution; 2) total protein load; 3) and LC. Our workflow simplifies the identification of these values.


Assuntos
Western Blotting/métodos , Proteínas/análise , Fluxo de Trabalho , Anticorpos/metabolismo , Humanos
3.
Hypertension ; 74(2): 313-322, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31230549

RESUMO

Human blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through an arginine regeneration pathway. We examined the role of the fumarase-NO pathway in the development of hypertension using genetically engineered rat models. Dahl salt-sensitive (SS) rats with heterozygous mutation of eNOS (endothelial NO synthase or Nos3; SS-Nos3+/-) were bred with SS rats with a hemizygous Fh transgene. SS-Nos3+/- rats without the Fh transgene (SS-Nos3+/-/Fh0/0) developed substantial hypertension with a mean arterial pressure of 134.2±3.7 mm Hg on a 0.4% NaCl diet and 178.0±3.5 mm Hg after 14 days on a 4% NaCl diet. Mean arterial pressure decreased remarkably to 123.1±1.4 mm Hg on 0.4% NaCl, and 143.3±1.5 mm Hg on 4% NaCl in SS-Nos3+/- rats with a Fh transgene (SS-Nos3+/-/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS-Nos3+/-/Fh0/1 rats compared with SS-Nos3+/-/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression in rats on the 0.4% NaCl diet. However, the decrease in NO metabolite in the renal outer medulla of SS-Nos3+/-/Fh0/0 rats on the 0.4% NaCl diet was reversed in SS-Nos3+/-/Fh0/1 rats, and levels of L-arginine, but not the other 12 amino acids analyzed, were significantly higher in SS-Nos3+/-/Fh0/1 rats than in SS-Nos3+/+/Fh0/0 rats. In conclusion, fumarase has potent effects in restoring NO production and blunting the development of hypertension attributable to eNOS haploinsufficiency.


Assuntos
Progressão da Doença , Fumarato Hidratase/genética , Haploinsuficiência/genética , Hipertensão/genética , Óxido Nítrico Sintase Tipo III/genética , Análise de Variância , Animais , Arginina/metabolismo , Biópsia por Agulha , Western Blotting/métodos , Cromatografia Líquida de Alta Pressão/métodos , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hipertensão/fisiopatologia , Imuno-Histoquímica , Masculino , Mutação/genética , Distribuição Aleatória , Ratos , Ratos Endogâmicos Dahl , Ratos Transgênicos , Urinálise/métodos
4.
Methods Enzymol ; 622: 293-307, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31155058

RESUMO

O-GlcNAcylation is a widespread posttranslational modification of intracellular proteins. Phenotypic and genetic experiments have established key roles for O-GlcNAc in development, mammalian cell survival, and several human diseases. However, the underlying mechanisms by which this modification alters biological pathways are still being discovered. An important part of this discovery process is the discovery of O-GlcNAcylated proteins, where chemical approaches have been particularly powerful. Here we describe how to combine one of these approaches, metabolic chemical reporters (MCRs), with bioorthogonal chemistry and Western blotting to identify potentially O-GlcNAcylated proteins.


Assuntos
Acetilglucosamina/análise , Western Blotting/métodos , Proteínas/química , Acetilglucosamina/metabolismo , Animais , Química Click/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Glicosilação , Humanos , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo
5.
BMJ Case Rep ; 12(4)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036741

RESUMO

The incidence of Lyme disease in the USA is 8 per 100 000 cases and 95% of those occur in the Northeastern region. Cardiac involvement occurs in only 1% of untreated patients. We describe the case of a 46-year-old man who presented with chest pressure, dyspnoea, palpitations and syncope. He presented initially with atrial fibrillation with rapid ventricular response, a rare manifestation of Lyme carditis. In another hospital presentation, he had varying degrees of atrioventricular block including Mobitz I second-degree heart block. After appropriate antibiotic treatment, he made a full recovery and his ECG normalised. The authors aim to urge physicians treating patients in endemic areas to consider Lyme carditis in the workup for patients with atrial fibrillation and unexplained heart block, as the associated atrioventricular nodal complications may be fatal.


Assuntos
Fibrilação Atrial/etiologia , Bloqueio Atrioventricular/etiologia , Doença de Lyme/diagnóstico , Miocardite/diagnóstico , Administração Intravenosa , Antibacterianos/uso terapêutico , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/fisiopatologia , Bloqueio Atrioventricular/diagnóstico , Bloqueio Atrioventricular/fisiopatologia , Western Blotting/métodos , Ceftriaxona/administração & dosagem , Ceftriaxona/uso terapêutico , Eletrocardiografia , Humanos , Doença de Lyme/complicações , Doença de Lyme/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Doenças Raras , Síncope/etiologia , Resultado do Tratamento
6.
Methods Mol Biol ; 1966: 79-99, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041740

RESUMO

Antibodies are invaluable biological tools that we can use to detect the presence, location, or alteration of nuclear receptors. However, antibodies frequently cross-react with other proteins and their performance can vary from batch to batch, from application to application and from lab to lab. When each lot of antibody is not thoroughly validated for each assay, each sample type, and each lab and user, antibody-based assays can lead to flawed interpretations and reproducibility problems. In this chapter, we describe a scheme for thorough antibody validation, suitable for nuclear receptors. The method is based on using highly characterized positive and negative controls assembled into a validation tissue microarray (TMA). Through correlation of immunohistochemical staining (IHC) and mRNA levels over multiple tissues, use of current public databases, and assessment of binding to intended and nonintended targets using western blotting (WB), immunoprecipitation (IP), and mass spectrometry (MS), we describe a path for thoroughly validation of antibodies.


Assuntos
Anticorpos Monoclonais/análise , Western Blotting/métodos , Imuno-Histoquímica/métodos , Receptores Citoplasmáticos e Nucleares/imunologia , Estudos de Validação como Assunto , Perfilação da Expressão Gênica , Humanos , Espectrometria de Massas , Reprodutibilidade dos Testes , Análise Serial de Tecidos/métodos
7.
Methods Mol Biol ; 1970: 341-353, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30963502

RESUMO

MicroRNAs are a class of small noncoding RNA involved in the mechanism of RNA silencing and regulation of gene expression at a posttranscriptional level. Recently, the discovery of their targets led to the understanding of the molecular role of these small molecules and their involvement in the pathogenesis of numerous diseases, including cancer. Not long ago, the improvement of several informatics tools for microRNA target prediction has supported the experimental research through the selection of potential mRNA as microRNA target candidates. Since the regulation mediated by microRNA affects gene expression at a posttranscriptional level, the analysis of the proteins encoded by the gene targets is essential in understanding the involvement of these small molecules in biological processes and their role in several diseases. In this chapter, we describe the experimental procedure of Western blotting applied to the validation of microRNA targets. Western blotting is one of the most common and largest know technique for protein analysis. This method, coupled with the luciferase assay, represents the standard procedure for the experimental confirmation of microRNA targeting.


Assuntos
Western Blotting/métodos , Biologia Computacional/métodos , Luciferases/metabolismo , MicroRNAs/genética , RNA Mensageiro/análise , Regulação da Expressão Gênica , Humanos , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estudos de Validação como Assunto
8.
Biotechniques ; 66(4): 171-178, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987443

RESUMO

Fractionation in Gram-negative bacteria is used to identify the subcellular localization of proteins, in particular the localization of exported recombinant proteins. The process of cell fractionation can be fraught with cross-contamination issues and often lacks supporting data for fraction purity. Here, we compare three periplasm extraction and two cell disruption techniques in different combinations to investigate which process gives uncontaminated compartments from Escherichia coli. From these data, a robust method named PureFrac was compiled that gives pure periplasmic fractions and a superior recovery of soluble cytoplasmic proteins. The process extracts periplasm using cold osmotic shock with magnesium, prior to sonication and ultracentrifugation to separate the cytoplasm from insoluble material. This method handles cells cultivated in various conditions and allows preparation of active proteins in their respective compartments.


Assuntos
Fracionamento Celular/métodos , Proteínas de Escherichia coli/análise , Escherichia coli/citologia , Periplasma/química , Proteínas Recombinantes/análise , Western Blotting/métodos , Temperatura Baixa , Escherichia coli/química , Pressão Osmótica
9.
Technol Cancer Res Treat ; 18: 1533033819828396, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30943868

RESUMO

To date, diagnosis of IDH1 mutation is based on DNA sequencing and immunohistochemistry, methods limited in terms of sensitivity and ease of use. Recently, the diagnosis of IDH1 mutation by real-time polymerase chain reaction was introduced as an alternative method. In this study, real-time polymerase chain reaction was validated as a tool for detection of IDH1 mutation, and expression levels were analyzed for correlation with course of the disease. A total of 113 tumor samples were obtained intraoperatively from 84 patients with glioma having a diagnosis of diffuse glioma (World Health Organization II), anaplastic glioma (World Health Organization III), secondary glioblastoma ± chemotherapy, primary glioblastoma ± chemotherapy (World Health Organization IV). Tumor samples were snap frozen and processed for sectioning and RNA and protein isolation. Presence of IDH1 mutation was determined by DNA sequencing. Hereafter, quantitative expression of IDH1 messenger RNA was assessed using real-time polymerase chain reaction with specific primers for IDH1 mutation and -wt; protein expression was verified by Western Blot analysis and immunohistochemistry. Additionally, 19 samples of low-grade glioma and their consecutive high-grade glioma were analyzed at different time points of the disease. IDH1 mutation was identified in 63% of samples by DNA sequencing. In correlation with the real-time polymerase chain reaction results, a cutoff value was determined. Above this threshold, sensitivity and specificity of real-time polymerase chain reaction in detecting IDH1 mutation were 98% and 94%, respectively. Quantitative analysis revealed that IDH1 mutation expression is upregulated in secondary glioblastoma (mean ± standard error of mean: 3.52 ± 0.55) compared to lower grade glioma (II = 1.54 ± 0.22; III = 1.67 ± 0.23). In contrast, IDH1 wt expression is upregulated in all glioma grades (concentration >0.1) compared to control brain tissue (0.007 ± 0.0016). Western Blot analysis showed a high concordance to both sequencing and real-time polymerase chain reaction results in qualitative analysis of IDH1 mutation status (specificity 100% and sensitivity 100%). Moreover, semiquantitative protein expression analysis also showed higher expression levels of mutated IDH1 in secondary glioblastoma. In our study, real-time polymerase chain reaction and Western Blot analysis were found to be highly efficient methods in detecting IDH1 mutation in glioma samples. As cost-effective and time-saving methods, real-time polymerase chain reaction and Western Blot analysis may therefore play an important role in IDH1 mutation analysis in the future. IDH1 mutation expression level was found to correlate with the course of disease to a certain extent. Yet, clinical factors as recurrent disease or prior radiochemotherapy did not alter IDH1 mutation expression level.


Assuntos
Western Blotting/métodos , Neoplasias Encefálicas/genética , Glioma/genética , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Mutação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto , Alelos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Feminino , Glioma/metabolismo , Glioma/patologia , Humanos , Masculino , Pessoa de Meia-Idade
10.
Bioanalysis ; 11(6): 471-483, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30892061

RESUMO

Aim: To develop and validate a reliable, robust and efficient assay to detect and quantify biologic compounds in vitro and in vivo during early stage of a biotherapeutic agent discovery. Methodology & results: An enrichment-free immunoassay method was developed to quantify a polyhistidine N- and FLAG C-terminally-tagged recombinant protein of ∼55 kDa. The target proteins were purified by a nickel-based matrix via tag affinity, followed by probing with biotinylated antitag antibody and subsequently detected by streptavidin-horseradish peroxidase conjugate using an automated capillary-based western system. Conclusion: A simple, highly sensitive and efficient immunoassay protocol was established to assess the in vitro stability and pharmacokinetic properties of propitious recombinant proteins in vivo in mouse to support early stage development of a biotherapeutic lead.


Assuntos
Epitopos/química , Imunoensaio/métodos , Proteínas Recombinantes/sangue , Animais , Biotinilação , Western Blotting/métodos , Histidina/química , Indicadores e Reagentes/química , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Níquel/química , Oligopeptídeos/sangue , Oligopeptídeos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Estreptavidina/química
11.
Biochemistry (Mosc) ; 84(1): 11-19, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30927521

RESUMO

Myosin II is the main molecular motor in the actomyosin-dependent motility in cells. Phosphorylation of the myosin regulatory light chain (RLC) at Ser19 is a prerequisite for smooth muscle/non-muscle myosin II activation and serves as a biochemical equivalent of myosin II activity. Simultaneous phosphorylation at Thr18 further promotes the myosin II ATPase activity. A number of methods have been developed to measure myosin RLC phosphorylation at Ser19 or di-phosphorylation at Thr18/Ser19. While these methods are straightforward and robust in myosin-rich muscle tissues, they demonstrate limited applicability in non-muscle cells that have low myosin II content and are usually available in lesser amounts than muscle tissue. Because of this, dynamic analysis of RLC phosphorylation in multiple samples of non-muscle cells is difficult and requires large number of cells. The use of phospho-specific antibodies increases detection sensitivity but allows estimation of only relative levels of RLC phosphorylation at specific residues, which makes it difficult to estimate the physiologic relevancy of the observed changes in RLC phosphorylation. To measure RLC phosphorylation in small amounts of non-muscle cells, we used external calibration standards of non-phosphorylated and in vitro phosphorylated RLC in standard SDS-PAGE and Western blot procedures with phospho-specific RLC antibodies. Here, we describe the method in detail and demonstrate its application for quantitative measurement of myosin RLC phosphorylation in endothelial cells in response to natural agonists (thrombin or insulin) and intact human platelets. We discuss the advantages and limitations of the proposed method vs other approaches for measuring myosin RLC phosphorylation in non-muscle cells.


Assuntos
Western Blotting/métodos , Cadeias Leves de Miosina/metabolismo , Fosforilação , Plaquetas , Eletroforese em Gel de Poliacrilamida/métodos , Células Endoteliais/metabolismo , Humanos , Insulina/farmacologia , Trombina/farmacologia
12.
Methods Mol Biol ; 1952: 157-191, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825174

RESUMO

A growing body of research demonstrates modulation of autophagy by a variety of matrix constituents, including decorin, endorepellin, and endostatin. These matrix proteins are both pro-autophagic and anti-angiogenic. Here, we detail a series of methods to monitor matrix-induced autophagy and its concurrent effects on angiogenesis. We first discuss cloning and purifying proteoglycan fragment and core proteins in the laboratory and review relevant techniques spanning from cell culture to treatment with these purified proteoglycans in vitro and ex vivo. Further, we cover protocols in monitoring autophagic progression via morphological and microscopic characterization, biochemical western blot analysis, and signaling pathway investigation. Downstream angiogenic effects using in vivo approaches are then discussed using wild-type mice and the GFP-LC3 transgenic mouse model. Finally, we explore matrix-induced mitophagy via monitoring changes in mitochondrial DNA and permeability.


Assuntos
Autofagia , Proteínas da Matriz Extracelular/metabolismo , Neovascularização Fisiológica , Proteoglicanas/metabolismo , Animais , Western Blotting/métodos , Clonagem Molecular/métodos , Proteínas da Matriz Extracelular/genética , Imunofluorescência/métodos , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Microscopia de Força Atômica/métodos , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Proteoglicanas/genética , Ratos , Transdução de Sinais
13.
Methods Mol Biol ; 1952: 233-244, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825179

RESUMO

Exosomes are small vesicles of endosomal origin secreted by most cell types. Recent studies have identified exosomes as important mediators of intercellular communication and as important source materials for many clinical applications, including minimal invasive disease diagnosis. Exosomes have been purified from in vitro cell culture supernatants by many different methods; however the most simple and reliable method involves purification by ultracentrifugation. This chapter describes a detailed protocol for isolating exosomes from cell culture-conditioned medium using ultracentrifugation and their characterization based upon size, number, and protein expression by several complementary methods such as transmission electron microscopy, nanoparticle tracking analysis, western blotting, and flow cytometry.


Assuntos
Exossomos/química , Exossomos/ultraestrutura , Ultracentrifugação/métodos , Animais , Western Blotting/métodos , Técnicas de Cultura de Células/métodos , Meios de Cultivo Condicionados/química , Citometria de Fluxo/métodos , Humanos , Microscopia Eletrônica de Transmissão/métodos , Proteínas/análise
14.
Methods Mol Biol ; 1922: 239-250, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838582

RESUMO

The organic material in developing dentin is 90% type I collagen and 10% non-collagenous proteins. The key to understanding dentin biomineralization is to study how these proteins collectively precipitate and organize hydroxyapatite crystals. The first step in characterizing the proteins within a mineralizing matrix is to efficiently extract and isolate the essential molecular participants and elucidate their structural and biochemical properties. In this study, we expanded previous approaches to develop an improved strategy for the extraction of extracellular matrix proteins from the dentin of developing teeth. Proteins in dentin powder were sequentially extracted in the order Tris-guanidine buffer, HCl-formic acid solution, acetic acid-NaCl solution, Tris-NaCl buffer, and a second Tris-guanidine buffer. Individual fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by gelatin or casein zymography, and by Western blot analysis using dentin sialoprotein (DSP)- or dentin glycoprotein (DGP)-specific antibodies. This approach was used to purify assorted porcine dentin non-collagenous proteins.


Assuntos
Western Blotting/métodos , Dentina/química , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas da Matriz Extracelular/isolamento & purificação , Glicoproteínas/isolamento & purificação , Fosfoproteínas/isolamento & purificação , Sialoglicoproteínas/isolamento & purificação , Dente/química , Ácido Acético/química , Animais , Dentina/crescimento & desenvolvimento , Formiatos/química , Guanidina/química , Suínos , Dente/crescimento & desenvolvimento , Trometamina/química
15.
Chem Commun (Camb) ; 55(24): 3473-3476, 2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30829351

RESUMO

Bruton's tyrosine kinase, an essential mediator of B cell receptor (BCR) signalling, has been validated as an effective therapeutic target in oncology. Development of chemical sensors capable of precise detection of its expression and function is of great importance for cancer diagnosis and therapy. Here, a dual-purpose probe, IB-4, was developed with excellent inhibitory activity (IC50 = 35 nM), and has been demonstrated to be suitable for simultaneous imaging endogenous BTK activity and studying its target engagement in live cells for the first time. The unique turn-on design endows the probe with excellent sensitivity and selectivity toward BTK under both in vitro and in situ settings, and can be further extended to other irreversible inhibitors for protein characterization, quantification and inhibition studies.


Assuntos
Tirosina Quinase da Agamaglobulinemia/análise , Corantes Fluorescentes/química , Western Blotting/métodos , Linhagem Celular , Sobrevivência Celular , Humanos , Células Jurkat , Simulação de Acoplamento Molecular , Imagem Óptica/métodos , Proteínas Recombinantes/análise
16.
Methods Enzymol ; 619: 319-336, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30910027

RESUMO

Autophagy is being studied intensively in Caenorhabditis elegans in the context of protein homeostasis and aging. However, in contrast to the yeast and mammalian autophagosomal membrane proteins Atg8 and LC3, lipidation of the C. elegans ortholog LGG-1 with phosphatidylethanolamine has rarely been investigated by western blotting. We attribute this shortcoming to technical problems with separating the nonlipidated from the lipidated LGG-1 protein by gel electrophoresis. Our new protocol for Western blot analysis is applicable for both the detection of transgenic and endogenous LGG-1 proteins and provides a quantifiable method to assess autophagic flux. As a proof of principle, we use this assay to analyze the role of the transcriptional master regulator HLH-30/TFEB in starvation-induced autophagy.


Assuntos
Autofagia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidiletanolaminas/metabolismo , Animais , Western Blotting/métodos , Caenorhabditis elegans/citologia , Eletroforese em Gel de Poliacrilamida/métodos , Proteínas de Membrana/metabolismo
17.
Methods Enzymol ; 618: 187-210, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30850052

RESUMO

Covalent modification of proteins with the small ubiquitin-related modifier (SUMO) is found in all eukaryotes and is involved in many important processes. SUMO attachment may change interaction properties, subcellular localization, or stability of a modified protein. Usually, only a small fraction of a protein is modified at a given time because sumoylation is a highly dynamic process. The sumoylated state of a protein is controlled by the activity of the sumoylation enzymes that promote either their mono- or poly-sumoylation (SUMO chain formation), by SUMO proteases that reverse these modifications, and by SUMO-targeted ubiquitin ligases (STUbL, ULS) that mediate their degradation by the proteasome. While some organisms, such as humans, express multiple isoforms, budding yeast SUMO is encoded by a single and essential gene termed SMT3. The analysis of the simpler SUMO system in budding yeast has been instrumental in the identification of enzymes acting on this modification and controlling its dynamics. Sumoylation of proteins changes dramatically during the cell division cycle and under various stress conditions. Here we summarize various approaches that employ Saccharomyces cerevisiae as a model system to study the dynamics of sumoylation and how it is controlled.


Assuntos
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Western Blotting/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Sumoilação , Ubiquitina/metabolismo
18.
Methods Mol Biol ; 1955: 135-146, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30868524

RESUMO

Trypanosoma cruzi, the protozoan agent of Chagas disease, has evolved an innovative metabolic pathway by which protective sialic acid (SA) residues are scavenged from host sialylglycoconjugates and transferred onto parasite surface mucin-like molecules (or surface glycoconjugates from host target cells) by means of a unique trans-sialidase (TS) enzyme. TS-induced changes in the glycoprotein sialylation profile of both parasite and host cells are crucial for the establishment of a persistent T. cruzi infection and for the development of Chagas disease-associated pathogenesis. In this chapter, we describe a novel metabolic labeling method developed in our labs that enables straightforward identification and molecular characterization of SA acceptors of the TS-catalyzed reaction.


Assuntos
Glicoproteínas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/fisiologia , Animais , Western Blotting/métodos , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Citometria de Fluxo/métodos , Imunofluorescência/métodos , Interações Hospedeiro-Parasita , Humanos , Redes e Vias Metabólicas , Coloração e Rotulagem/métodos , Trypanosoma cruzi/enzimologia
19.
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
20.
Methods Enzymol ; 617: 443-461, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30784412

RESUMO

New biological properties can stem from the freedom to link, multimerize, or multiplex protein building blocks. The peptide SpyTag on one protein irreversibly reacts with SpyCatcher on another protein, through spontaneous isopeptide bond formation. Reaction is specific in a wide range of cellular environments and all components are genetically encoded, making this chemistry accessible to molecular biologists. SpyTag/SpyCatcher has been widely used for enzyme immobilization, colocalization of different enzymatic activities, and increasing enzyme resilience. Here we present routes and advice for efficient design, expression, and purification of SpyTag/SpyCatcher constructs in bacterial and eukaryotic environments, including the latest 002 variants, and how to analyze reaction efficiency. The SpyInfo webpage collates the different publications and patents using SpyTag/SpyCatcher, while the SpyBank database lists their sequences and expression routes. The ability of SpyTag/SpyCatcher to react in a broad range of situations creates diverse opportunities for augmenting the function of enzymes and other biomolecules.


Assuntos
Peptídeos/genética , Western Blotting/métodos , Bases de Dados de Proteínas , Eletroforese em Gel de Poliacrilamida/métodos , Escherichia coli/genética , Expressão Gênica , Células HEK293 , Humanos , Peptídeos/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
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