RESUMO
Phosphorylation is a major constituent of the CTD code, which describes the set of post-translational modifications on 52 repeats of a YSPTSPS consensus heptad that orchestrates the binding of regulatory proteins to the C-terminal domain (CTD) of RNA polymerase II. Phospho-specific antibodies are used to detect CTD phosphorylation patterns. However, their recognition repertoire is underexplored due to limitations in the synthesis of long multiphosphorylated peptides. Herein, we describe the development of a synthesis strategy that provides access to multiphosphorylated CTD peptides in high purity without HPLC purification for immobilization onto microtiter plates. Native chemical ligation was used to assemble 12 heptad repeats in various phosphoforms. The synthesis of >60 CTD peptides, 48-90 amino acids in length and containing up to 6 phosphosites, enabled a detailed and rapid analysis of the binding characteristics of different anti-pSer2 antibodies. The three antibodies tested showed positional selectivity with marked differences in the affinity of the antibodies for pSer2-containing peptides. Furthermore, the length of the phosphopeptides allowed a systematic analysis of the multivalent chelate-type interactions. The absence of multivalency-induced binding enhancements is probably due to the high flexibility of the CTD scaffold. The effect of clustered phosphorylation proved to be more complex. Recognition of pSer2 by anti-pSer2-antibodies can be prevented and, perhaps surprisingly, enhanced by the phosphorylation of "bystander" amino acids in the vicinity. The results have relevance for functional analysis of the CTD in cell biological experiments.
Assuntos
Anticorpos Monoclonais , Anticorpos Fosfo-Específicos , Fosfopeptídeos , RNA Polimerase II , Domínios Proteicos/imunologia , RNA Polimerase II/química , RNA Polimerase II/imunologia , Anticorpos Monoclonais/química , Anticorpos Fosfo-Específicos/química , Fosfopeptídeos/síntese química , Fosfopeptídeos/química , Fosfopeptídeos/imunologia , Ligação Proteica , Sítios de Ligação , Sequência de Aminoácidos , Biblioteca de PeptídeosRESUMO
Bub1 is a conserved mitotic kinase involved in signaling of the spindle assembly checkpoint. Multiple phosphorylation sites on Bub1 have been characterized, yet it is challenging to understand the interplay between the multiple phosphorylation sites due to the limited availability of phosphospecific antibodies. In addition, phosphoregulation of Bub1 in Schizosaccharomyces pombe is poorly understood. Here we report the identification of a new Mph1/Mps1-mediated phosphorylation site, i.e., Ser532, of Bub1 in Schizosaccharomyces pombe. A phosphospecific antibody against phosphorylated Bub1-Ser532 was developed. Using the phosphospecific antibody, we demonstrated that phosphorylation of Bub1-Ser352 was mediated specifically by Mph1/Mps1 and took place during early mitosis. Moreover, live-cell microscopy showed that inhibition of the phosphorylation of Bub1 at Ser532 impaired the localization of Bub1, Mad1, and Mad2 to the kinetochore. In addition, inhibition of the phosphorylation of Bub1 at Ser532 caused anaphase B lagging chromosomes. Hence, our study constitutes a model in which Mph1/Mps1-mediated phosphorylation of fission yeast Bub1 promotes proper kinetochore localization of Bub1 and faithful chromosome segregation.
Assuntos
Segregação de Cromossomos , Cinetocoros , Proteínas Serina-Treonina Quinases , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Transdução de Sinais , Anáfase , Anticorpos Fosfo-Específicos/imunologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Mitose , Fosforilação , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/imunologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Fuso Acromático/metabolismoRESUMO
Spatial proteomics has recently garnered significant interest, as it offers to provide unprecedented insight into biological processes in both health and disease, by connecting protein expression patterns from the subcellular level to the tissue or even organism level. These high-content approaches generally rely on a high degree of multiplexing, whereby multiple proteins can be detected simultaneously. The most versatile multiplexing approaches utilize antibodies to confer specificity for various intracellular proteins of interest. Therefore, these methods must be able to differentiate many antibodies at once. In this chapter, we describe a simple and rapid approach to labeling antibodies with distinct epitope tags in a site-specific manner. This allows multiple antibodies, even from the same host species, to be uniquely identified and detected and offers a simple approach for spatial proteomic applications.
Assuntos
Anticorpos , Proteômica , Epitopos/metabolismo , Anticorpos Fosfo-Específicos , Anticorpos/metabolismo , ProteínasRESUMO
Cancer is a fatal disease worldwide. Each year ten million people are diagnosed around the world, and more than half of patients eventually die from it in many countries. A majority of cancer remains asymptomatic in the earlier stages, with specific symptoms appearing in the advanced stages when the chances of adequate treatment are low. Cancer screening is generally executed by different imaging techniques like ultrasonography (USG), mammography, CT-scan, and magnetic resonance imaging (MRI). Imaging techniques, however, fail to distinguish between cancerous and non-cancerous cells for early diagnosis. To confirm the imaging result, solid and liquid biopsies are done which have certain limitations such as invasive (in case of solid biopsy) or missed early diagnosis due to extremely low concentrations of circulating tumor DNA (in case of liquid biopsy). Therefore, it is essential to detect certain biomarkers by a noninvasive approach. One approach is a proteomic or glycoproteomic study which mostly identifies proteins and glycoproteins present in tissues and serum. Some of these studies are approved by the Food and Drug Administration (FDA). Another non-expensive and comparatively easier method to detect glycoprotein biomarkers is by ELISA, which uses lectins of diverse specificities. Several of the FDA approved proteins used as cancer biomarkers do not show optimal sensitivities for precise diagnosis of the diseases. In this regard, expression of phosphoproteins is associated with a more specific stage of a particular disease with high sensitivity and specificity. In this review, we discuss the expression of different serum phosphoproteins in various cancers. These phosphoproteins are detected either by phosphoprotein enrichment by immunoprecipitation using phosphospecific antibody and metal oxide affinity chromatography followed by LC-MS/MS or by 2D gel electrophoresis followed by MALDI-ToF/MS analysis. The updated knowledge on phosphorylated proteins in clinical samples from various cancer patients would help to develop these serum phophoproteins as potential diagnostic/prognostic biomarkers of cancer.
Assuntos
DNA Tumoral Circulante , Neoplasias , Humanos , Cromatografia Líquida , Proteômica/métodos , Espectrometria de Massas em Tandem , Anticorpos Fosfo-Específicos , Detecção Precoce de Câncer , Biomarcadores Tumorais , Fosfoproteínas/análise , Neoplasias/diagnóstico , Glicoproteínas , Lectinas , ÓxidosRESUMO
The cancer phenotype is usually characterized by deregulated activity of a variety of cellular kinases, with consequent abnormal hyper-phosphorylation of their target proteins. Therefore, antibodies that allow the detection of phosphorylated versions of proteins have become important tools both preclinically in molecular cancer research, and at the clinical level by serving as tools in pathological analyses of tumors. In order to ensure reliable results, validation of the phospho-specificity of these antibodies is extremely important, since this ensures that they are indeed able to discriminate between the phosphorylated and unphosphorylated versions of the protein of interest, specifically recognizing the phosphorylated variant. A recommended validation approach consists in dephosphorylating the target protein and assessing if such dephosphorylation abrogates antigen immunoreactivity when using the phospho-specific antibody. In this chapter, we describe a protocol to validate the specificity of a phospho-specific antibody that recognizes a phosphorylated variant of the Retinoblastoma (Rb) protein in lung cancer cell lines. The protocol consists in the dephosphorylation of the Rb-containing protein lysates by treating them with bovine intestinal phosphatase, followed by assessment of the dephosphorylation by immunoblot.
Assuntos
Anticorpos Antineoplásicos/química , Anticorpos Fosfo-Específicos/química , Immunoblotting , Neoplasias Pulmonares/metabolismo , Fosfoproteínas/metabolismo , Proteína do Retinoblastoma/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/patologiaRESUMO
The cytoplasmic region of the γ chain of the high-affinity receptor for IgE (FcεRI) contains a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of the two tyrosine residues (N-terminal Y47 and C-terminal Y58) in the ITAM sequence is crucial for the recruitment and activation of Syk, a cytoplasmic tyrosine kinase with central signaling roles in mast cells. Using a reconstitution system in which individual tyrosine-to-phenylalanine substituted γ chains were expressed in γ-chain-deficient mast cells, we previously reported differential dephosphorylation of these tyrosines. Herein, we developed monoclonal antibodies highly specific to the phosphorylated Y47 and Y58 residues, which enables monitoring their phosphorylation under more physiological conditions. Using these antibodies, preferential dephosphorylation of Y58 following FcεRI stimulation was confirmed. Furthermore, Y58 is potentially more susceptible to phosphorylation than is Y47. Consistent with this, an in vitro kinase assay using these phospho-specific antibodies demonstrated that the Src family kinase Lyn, which is primarily responsible for ITAM phosphorylation, phosphorylates Y58 more efficiently than Y47. These results indicate that Y58 is more susceptible to dephosphorylation and phosphorylation than is Y47. Because a phosphate group on Y58 is more important for Syk binding than is a phosphate group on Y47, the preferential phosphorylation and dephosphorylation of Y58 may contribute to the fine tuning of Syk activity by promoting rapid recruitment and reducing excessive activation.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Fosfo-Específicos/metabolismo , Motivo de Ativação do Imunorreceptor Baseado em Tirosina , Mastócitos/imunologia , Receptores de IgG/metabolismo , Quinase Syk/metabolismo , Tirosina/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Fosfo-Específicos/imunologia , Células Cultivadas , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL , Fosforilação , Receptores de IgG/química , Transdução de Sinais , Tirosina/químicaRESUMO
Antibodies raised against defined phosphorylation sites of the microtubule-associated protein tau are widely used in scientific research and being applied in clinical assays. However, recent studies have revealed an alarming degree of non-specific binding found in these antibodies. In order to quantify and compare the specificity phospho-tau antibodies and other post-translational modification site-specific antibodies in general, a measure of specificity is urgently needed. Here, we report a robust flow cytometry assay using human embryonic kidney cells that enables the determination of a specificity parameter termed Φ, which measures the fraction of non-specific signal in antibody binding. We validate our assay using anti-tau antibodies with known specificity profiles, and apply it to measure the specificity of seven widely used phospho-tau antibodies (AT270, AT8, AT100, AT180, PHF-6, TG-3, and PHF-1) among others. We successfully determined the Φ values for all antibodies except AT100, which did not show detectable binding in our assay. Our results show that antibodies AT8, AT180, PHF-6, TG-3, and PHF-1 have Φ values near 1, which indicates no detectable non-specific binding. AT270 showed Φ value around 0.8, meaning that approximately 20% of the binding signal originates from non-specific binding. Further analyses using immunocytochemistry and western blotting confirmed the presence of non-specific binding of AT270 to non-tau proteins found in human embryonic kidney cells and the mouse hippocampus. We anticipate that the quantitative approach and parameter introduced here will be widely adopted as a standard for reporting the specificity for phospho-tau antibodies, and potentially for post-translational modification targeting antibodies in general. Cover Image for this issue: doi: 10.1111/jnc.14727.
Assuntos
Anticorpos Fosfo-Específicos/imunologia , Especificidade de Anticorpos/imunologia , Proteínas tau/imunologia , Animais , Citometria de Fluxo/métodos , Corantes Fluorescentes , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas de Fluorescência Verde , Células HEK293 , Hipocampo/química , Humanos , Imuno-Histoquímica , Rim/química , Camundongos , Fosforilação , TransfecçãoRESUMO
Post-translational modifications, such as the phosphorylation of tyrosines, are often the initiation step for intracellular signaling cascades. Pan-reactive antibodies against modified amino acids (e.g., anti-phosphotyrosine), which are often used to assay these changes, require isolation of the specific protein prior to analysis and do not identify the specific residue that has been modified (in the case that multiple amino acids have been modified). Phosphorylation state-specific antibodies (PSSAs) developed to recognize post-translational modifications within a specific amino acid sequence can be used to study the timeline of modifications during a signal cascade. We used the FcεRI receptor as a model system to develop and characterize high-affinity PSSAs using phage and yeast display technologies. We selected three ß-subunit antibodies that recognized: 1) phosphorylation of tyrosines Y218 or Y224; 2) phosphorylation of the Y228 tyrosine; and 3) phosphorylation of all three tyrosines. We used these antibodies to study the receptor activation timeline of FcεR1 in rat basophilic leukemia cells (RBL-2H3) upon stimulation with DNP24-BSA. We also selected an antibody recognizing the N-terminal phosphorylation site of the γ-subunit (Y65) of the receptor and applied this antibody to evaluate receptor activation. Recognition patterns of these antibodies show different timelines for phosphorylation of tyrosines in both ß and γ subunits. Our methodology provides a strategy to select antibodies specific to post-translational modifications and provides new reagents to study mast cell activation by the high-affinity IgE receptor, FcεRI.
Assuntos
Anticorpos Fosfo-Específicos/metabolismo , Anticorpos/isolamento & purificação , Basófilos/fisiologia , Receptores de IgE/metabolismo , Leveduras/fisiologia , Animais , Anticorpos Fosfo-Específicos/química , Linhagem Celular , Técnicas de Visualização da Superfície Celular , Fosforilação , Processamento de Proteína Pós-Traducional , Ratos , Tirosina/imunologia , Tirosina/metabolismoRESUMO
Hypothalamic AMPK plays a key role in the control of energy homeostasis by regulating energy intake and energy expenditure, particularly modulating brown adipose tissue (BAT) thermogenesis. The function of AMPK can be assayed by analyzing its phosphorylated protein levels in tissues, since AMPK is activated when it is phosphorylated at Thr-172. Here, we describe a method to obtain hypothalamic (nuclei-specific) protein extracts and the suitable conditions to assay AMPK phosphorylation by Western blotting.
Assuntos
Proteínas Quinases Ativadas por AMP/análise , Ativação Enzimática/efeitos dos fármacos , Ensaios Enzimáticos/métodos , Hipotálamo/metabolismo , Isoenzimas/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/imunologia , Proteínas Quinases Ativadas por AMP/metabolismo , Adenoviridae/genética , Animais , Anticorpos Fosfo-Específicos/imunologia , Ativação Enzimática/genética , Ativadores de Enzimas/farmacologia , Ensaios Enzimáticos/instrumentação , Inibidores Enzimáticos/farmacologia , Vetores Genéticos/genética , Isoenzimas/genética , Isoenzimas/imunologia , Camundongos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fosforilação/imunologia , Ratos , Técnicas Estereotáxicas/instrumentação , Treonina/imunologia , Treonina/metabolismoRESUMO
The addition of phosphate groups to substrates allows protein kinases to regulate a myriad of biological processes, and contextual analysis of protein-bound phosphate is important for understanding how kinases contribute to physiology and disease. Leucine-rich repeat kinase 2 (LRRK2) is a Ser/Thr kinase linked to familial and sporadic cases of Parkinson's disease (PD). Recent work established that multiple Rab GTPases are physiological substrates of LRRK2, with Rab10 in particular emerging as a human substrate whose site-specific phosphorylation mirrors hyperactive LRRK2 lesions associated with PD. However, current assays to quantify Rab10 phosphorylation are expensive, time-consuming and technically challenging. In back-to-back studies reported in the Biochemical Journal, Alessi and colleagues teamed up with clinical colleagues and collaborators at the Michael J. Fox Foundation (MJFF) for Parkinson's research to develop, and validate, a panel of exquisitely sensitive phospho-specific Rab antibodies. Of particular interest, the monoclonal antibody-designated MJFF-pRAB10 detects phosphorylated Rab 10 on Thr73 in a variety of cells, brain extracts, PD-derived samples and human neutrophils, the latter representing a previously unrecognised biological resource for LRRK2 signalling analysis. In the future, these antibodies could become universal resources in the fight to understand and quantify connections between LRRK2 and Rab proteins, including those associated with clinical PD.
Assuntos
Fenômenos Biológicos , Doença de Parkinson , Anticorpos Fosfo-Específicos , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Transdução de SinaisRESUMO
We investigated UV-induced signalling in an ex vivo skin organ culture model using phospho-antibody array. Phosphorylation modulations were analysed in time-course experiments following exposure to solar-simulated UV and validated by Western blot analyses. We found that UV induced P-p38 and its substrates, P-ERK1/2 and P-AKT, which were previously shown to be upregulated by UV in cultured keratinocytes and in vivo human skin. This indicates that phospho-antibody array applied to ex vivo skin organ culture is a relevant experimental system to investigate signalling events following perturbations. As the identified proteins are components of pathways implicated in skin tumorigenesis, UV-exposed skin organ culture model could be used to investigate the effect on these pathways of NMSC cancer drug candidates. In addition, we found that phospho-HCK is induced upon UV exposure, producing a new candidate for future studies investigating its role in the skin response to UV and UV-induced carcinogenesis.
Assuntos
Anticorpos Fosfo-Específicos/análise , Técnicas de Cultura de Órgãos , Análise Serial de Proteínas , Transdução de Sinais/efeitos da radiação , Pele/efeitos da radiação , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Raios UltravioletaRESUMO
Mutations that activate the LRRK2 (leucine-rich repeat protein kinase 2) protein kinase predispose to Parkinson's disease, suggesting that LRRK2 inhibitors might have therapeutic benefit. Recent work has revealed that LRRK2 phosphorylates a subgroup of 14 Rab proteins, including Rab10, at a specific residue located at the centre of its effector-binding switch-II motif. In the present study, we analyse the selectivity and sensitivity of polyclonal and monoclonal phospho-specific antibodies raised against nine different LRRK2-phosphorylated Rab proteins (Rab3A/3B/3C/3D, Rab5A/5B/5C, Rab8A/8B, Rab10, Rab12, Rab29[T71], Rab29[S72], Rab35 and Rab43). We identify rabbit monoclonal phospho-specific antibodies (MJFF-pRAB10) that are exquisitely selective for LRRK2-phosphorylated Rab10, detecting endogenous phosphorylated Rab10 in all analysed cell lines and tissues, including human brain cingulate cortex. We demonstrate that the MJFF-pRAB10 antibodies can be deployed to assess enhanced Rab10 phosphorylation resulting from pathogenic (R1441C/G or G2019S) LRRK2 knock-in mutations as well as the impact of LRRK2 inhibitor treatment. We also identify rabbit monoclonal antibodies displaying broad specificity (MJFF-pRAB8) that can be utilised to assess LRRK2-controlled phosphorylation of a range of endogenous Rab proteins, including Rab8A, Rab10 and Rab35. The antibodies described in the present study will help with the assessment of LRRK2 activity and examination of which Rab proteins are phosphorylated in vivo These antibodies could also be used to assess the impact of LRRK2 inhibitors in future clinical trials.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Fosfo-Específicos/biossíntese , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação , Proteínas rab de Ligação ao GTP/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Fosfo-Específicos/química , Anticorpos Fosfo-Específicos/isolamento & purificação , Especificidade de Anticorpos , Regulação da Expressão Gênica , Predisposição Genética para Doença , Giro do Cíngulo/enzimologia , Giro do Cíngulo/fisiopatologia , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Camundongos , Família Multigênica , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Fosforilação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Coelhos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant, homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast, the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity, we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação , Neutrófilos/imunologia , Doença de Parkinson/genética , Proteínas rab de Ligação ao GTP/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Fosfo-Específicos/química , Anticorpos Fosfo-Específicos/isolamento & purificação , Especificidade de Anticorpos , Estudos de Casos e Controles , Ensaios Clínicos como Assunto , Ensaios Enzimáticos , Epitopos/química , Epitopos/imunologia , Regulação da Expressão Gênica , Predisposição Genética para Doença , Giro do Cíngulo/imunologia , Giro do Cíngulo/fisiopatologia , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Neutrófilos/patologia , Doença de Parkinson/enzimologia , Doença de Parkinson/imunologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Coelhos , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/imunologiaRESUMO
The dopamine transporter (DAT), which mediates the inactivation of released dopamine through its reuptake, is the primary molecular target for the actions of psychostimulants. An increasing number of studies support an essential role for phosphorylation of serines (Ser) in the distal amino (N) terminus of DAT in regulating its function. Still, the molecular details of the regulation of phosphorylation and its impact on function are not fully understood. To address this, we have developed and characterized two distinct phospho-antibodies that recognize human DAT when it is phosphorylated at Ser7 or Ser12. Our data show that treatment of cells with phorbol 12-myristate 13-acetate (PMA), amphetamine (AMPH) or okadaic acid (OA) leads to an increase in the phosphorylation of DAT at both residues and that these responses are dependent on the activity of protein kinase C. We also show that AMPH-induced and OA-induced phosphorylation of DAT are dependent on Ca2+/calmodulin-dependent protein kinase α. Our data further suggest that the lipid raft localization of DAT is necessary for efficient N-terminal phosphorylation and for the associated behavioral effects of AMPH, demonstrating the potential of these novel antibodies as powerful tools to study DAT regulation and function in vivo.
Assuntos
Anticorpos Fosfo-Específicos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Animais , Especificidade de Anticorpos , Proteínas da Membrana Plasmática de Transporte de Dopamina/análise , Drosophila , Humanos , Camundongos , FosforilaçãoRESUMO
Forty-eight surface sediments were collected from three mangrove wetlands in the Pearl River Estuary (PRE) of South China to investigate the distribution of organophosphorus flame retardants (OPFRs) and the relationship between OPFRs and microbial community structure determined by phospholipid fatty acid. Concentrations of ΣOPFRs in mangrove sediments of the PRE ranged from 13.2 to 377.1 ng g-1 dry weight. Levels of ΣOPFRs in mangrove sediments from Shenzhen and Guangzhou were significantly higher than those from Zhuhai, indicating that OPFRs were linked to industrialization and urbanization. Tris(chloropropyl)phosphate was the predominant profile of OPFRs in mangrove sediments from Shenzhen (38.9%) and Guangzhou (35.0%), while the composition profile of OPFRs in mangrove sediments from Zhuhai was dominated by tris(2-chloroethyl) phosphate (25.5%). The mass inventories of OPFRs in the mangrove sediments of Guangzhou, Zhuhai and Shenzhen were 439.5, 133.5 and 662.3 ng cm-2, respectively. Redundancy analysis revealed that OPFRs induced a shift in the structure of mangrove sediment microbial community and the variations were significantly correlated with tris(1,3-dichloro-2-propyl)phosphate and tris(2-butoxyethyl) phosphate.
Assuntos
Retardadores de Chama , Áreas Alagadas , Anticorpos Fosfo-Específicos/análise , China , Estuários , Retardadores de Chama/análise , Sedimentos Geológicos/química , Sedimentos Geológicos/microbiologia , Fenômenos Microbiológicos , Organofosfatos/análise , Compostos Organofosforados/análiseRESUMO
A complex composed of goat anti-rabbit secondary antibody conjugated to a polymer coated with horseradish peroxidase (HRP) molecules was used to develop rapid and highly sensitive immunostaining protocol for the detection of phosphorylated p27/Kip1 (T157) in human tissues. This polymer-HRP complex produced much better sensitivity detection compared to conventional biotin-streptavidin-HRP chemistry. Using polymer-HRP made it possible to reduce primary antibody concentration, eliminate some incubation steps such as avidin-biotin blocking and incubation with separate biotinylated secondary antibodies, and shorten the incubation time with primary antibody. Specificity of the detection was confirmed by eliminating labeling after treating tissues with lambda phosphatase to remove phosphate groups from p27/Kip1. Secondary antibodies conjugated to polymer-HRP is a reagent of choice in both research and diagnostic pathology allowing detecting low abundant and weakly expressed tissue targets.
Assuntos
Anticorpos Fosfo-Específicos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Peroxidase do Rábano Silvestre , Imunoconjugados , Imuno-Histoquímica/métodos , Fosfoproteínas/metabolismo , Anticorpos Fosfo-Específicos/imunologia , Especificidade de Anticorpos/imunologia , Inibidor de Quinase Dependente de Ciclina p27/imunologia , Peroxidase do Rábano Silvestre/química , Humanos , Fosfoproteínas/imunologia , Fosforilação , Sensibilidade e EspecificidadeRESUMO
Phospho-specific primary antibodies are used in immunohistochemistry (IHC) to detect phosphorylated sequences in proteins, in some cases they may also cross-react with non- or de-phosphorylated sequences. To rule out nonspecific staining, and to determine that the staining pattern is specific it is necessary to employ a so-called absorption control: phospho-specific primary antibodies are first incubated with phospho-peptide immunogen to block antibody binding sites, and this mixture is applied to tissue sections. If the antibody pre-blocked with cognate immunogen does not produce tissue staining, then the antibody is considered specific. However, if the staining does occur, it indicates that the antibody is nonspecific. The drawback of doing absorption by mixing the peptide with the antibody is that in solution such peptide-antibody complexes can dissociate unblocking the antibody which becomes capable of binding to cell and tissue targets, producing unwanted staining. To overcome this problem, we have developed a simple absorption control technique allowing for efficient blocking of phospho-specific antibodies with phospho-peptides immobilized on magnetic beads. This technique allows for sequestration of peptide-antibody complex from the incubation mixture eliminating the risk of un-blocking primary antibodies via their dissociation from the blocking peptide.
Assuntos
Imunofluorescência , Imuno-Histoquímica/métodos , Técnicas de Imunoadsorção , Fosfopeptídeos/metabolismo , Animais , Anticorpos Fosfo-Específicos/imunologia , Camundongos , Microscopia Confocal , Células NIH 3T3 , Fosfopeptídeos/imunologiaRESUMO
Numerous bacterial pathogens employ specialized protein secretion machineries to directly inject anti-host proteins, termed effector proteins, into eukaryotic cells. Effector proteins carrying small phosphorylatable tags can be used to detect and quantify effector protein injection. Here, we describe the use of the ELK- and GSK-tags to detect the translocation of the Y. pestis YopE effector protein into RAW 264.7 macrophage-like cells using immunoblot analysis with phospho-specific antibodies.
Assuntos
Anticorpos Fosfo-Específicos , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Epitopos , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Fosfo-Específicos/imunologia , Proteínas de Bactérias/química , Western Blotting , Linhagem Celular , Epitopos/química , Epitopos/imunologia , Ordem dos Genes , Camundongos , Fosforilação , Plasmídeos/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Yersinia pestis/imunologia , Yersinia pestis/metabolismoRESUMO
Peptides (usually 10-20 amino acid residues in length) can be used as effectively as proteins in raising antibodies producing both polyclonal and monoclonal antibodies routinely with titers higher than 20,000. Peptide antigens do not function as immunogens unless they are conjugated to proteins. Production of high quality antipeptide antibodies is dependent upon peptide sequence selection, the success of peptide synthesis, peptide-carrier protein conjugation, the humoral immune response in the host animal, the adjuvant used, the peptide dose administered, the injection method, and the purification of the antibody. Peptide sequence selection is probably the most critical step in the production of antipeptide antibodies. Although the process for designing peptide antigens is not exact, several guidelines and computational B-cell epitope prediction methods can help maximize the likelihood of producing antipeptide antibodies that recognize the protein. Antibodies raised by peptides have become essential tools in life science research. Virtually all phospho-specific antibodies are now produced using phosphopeptides as antigens. Typically, 5-20 mg of peptide is enough for antipeptide antibody production. It takes 3 months to produce a polyclonal antipeptide antibody in rabbits that yields ~100 mL of serum which corresponds to ~8-10 mg of the specific antibody after affinity purification using a peptide column.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Fosfo-Específicos/biossíntese , Antígenos/química , Epitopos de Linfócito B/química , Peptídeos/síntese química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Fosfo-Específicos/isolamento & purificação , Especificidade de Anticorpos , Antígenos/imunologia , Linfócitos B/citologia , Linfócitos B/imunologia , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/imunologia , Hemocianinas/química , Hemocianinas/imunologia , Interações Hidrofóbicas e Hidrofílicas , Peptídeos/administração & dosagem , Peptídeos/imunologia , Coelhos , SoftwareRESUMO
Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3'UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future.