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Proximity ligation assay

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https://www.readbyqxmd.com/read/28547925/blockade-of-the-dissociation-of-nmmhc-iia-from-tnfr2-by-d39-a-saponin-from-liriope-muscari-suppresses-tissue-factor-expression-and-venous-thrombosis
#1
Ke-Feng Zhai, Jin-Rong Zheng, You-Mei Tang, Fang Li, Yan-Ni Lv, Yuan-Yuan Zhang, Zhen Gao, Jin Qi, Bo-Yang Yu, Jun-Ping Kou
BACKGROUND AND PURPOSE: Non-muscular myosin heavy chain IIA (NMMHC IIA) has been demonstrated to play a key role in tissue factor (TF) expression and venous thrombosis. However, whether natural products could inhibit thrombosis via NMMHC IIA remains unclear. In this study, we identified that a representative natural saponin D39 from Liriope muscari showed anti-thrombotic activity by targeting NMMHC IIA. EXPERIMENTAL APPROACH: TF expression and activity in endothelial cells were analysed in vitro by western blot and simplified chromogenic assays...
May 26, 2017: British Journal of Pharmacology
https://www.readbyqxmd.com/read/28522883/in-situ-evaluation-of-estrogen-receptor-dimers-in-breast-carcinoma-cells-visualization-of-protein-protein-interactions
#2
Erina Iwabuchi, Yasuhiro Miki, Katsuhiko Ono, Yoshiaki Onodera, Hironobu Sasano
The estrogen receptor (ER) functions as a dimer and is involved in several different biological functions. However ER dimeric proteins have not been identified by in situ methodologies. Structured illumination microscopy (SIM) has been recently developed, which enabled the localization of protein and protein interaction. Therefore, in this study, we firstly demonstrated that ERs formed both homodimers and heterodimers in breast carcinoma cell lines using Nikon's SIM (N-SIM). ERα/α homodimers were detected in the nuclei of both ERα-positive MCF-7 and T-47D cells; 23...
April 27, 2017: Acta Histochemica et Cytochemica
https://www.readbyqxmd.com/read/28500295/angiotensin-ii-type-1-adenosine-a-2a-receptor-oligomers-a-novel-target-for-tardive-dyskinesia
#3
Paulo A de Oliveira, James A R Dalton, Marc López-Cano, Adrià Ricarte, Xavier Morató, Filipe C Matheus, Andréia S Cunha, Christa E Müller, Reinaldo N Takahashi, Víctor Fernández-Dueñas, Jesús Giraldo, Rui D Prediger, Francisco Ciruela
Tardive dyskinesia (TD) is a serious motor side effect that may appear after long-term treatment with neuroleptics and mostly mediated by dopamine D2 receptors (D2Rs). Striatal D2R functioning may be finely regulated by either adenosine A2A receptor (A2AR) or angiotensin receptor type 1 (AT1R) through putative receptor heteromers. Here, we examined whether A2AR and AT1R may oligomerize in the striatum to synergistically modulate dopaminergic transmission. First, by using bioluminescence resonance energy transfer, we demonstrated a physical AT1R-A2AR interaction in cultured cells...
May 12, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28485577/site-selective-conjugation-of-native-proteins-with-dna
#4
Julie B Trads, Thomas Tørring, Kurt V Gothelf
Conjugation of DNA to proteins is increasingly used in academia and industry to provide proteins with tags for identification or handles for hybridization to other DNA strands. Assay technologies such as immuno-PCR and proximity ligation and the imaging technology DNA-PAINT require DNA-protein conjugates. In DNA nanotechnology, the DNA handle is exploited to precisely position proteins by self-assembly. For these applications, site-selective conjugation is almost always desired because fully functional proteins are required to maintain the specificity of antibodies and the activity of enzymes...
May 9, 2017: Accounts of Chemical Research
https://www.readbyqxmd.com/read/28475172/keratins-regulate-colonic-epithelial-cell-differentiation-through-the-notch1-signalling-pathway
#5
Iris A K Lähdeniemi, Julia O Misiorek, Christian J M Antila, Sebastian K-J Landor, Carl-Gustaf A Stenvall, Lina E Fortelius, Linda K Bergström, Cecilia Sahlgren, Diana M Toivola
Keratins (K) are intermediate filament proteins important in stress protection and mechanical support of epithelial tissues. K8, K18 and K19 are the main colonic keratins, and K8-knockout (K8(-/-)) mice display a keratin dose-dependent hyperproliferation of colonic crypts and a colitis-phenotype. However, the impact of the loss of K8 on intestinal cell differentiation has so far been unknown. Here we show that K8 regulates Notch1 signalling activity and differentiation in the epithelium of the large intestine...
June 2017: Cell Death and Differentiation
https://www.readbyqxmd.com/read/28473697/highly-sensitive-and-specific-protein-detection-via-combined-capillary-isoelectric-focusing-and-proximity-ligation
#6
Narendra Padhan, Junhong Yan, Annegret Boge, Elaine Scrivener, Helgi Birgisson, Agata Zieba, Mats Gullberg, Masood Kamali-Moghaddam, Lena Claesson-Welsh, Ulf Landegren
Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms...
May 4, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28471195/-application-of-pla-method-for-detection-of-p53-p63-p73-complexes-in-situ-in-tumour-cells-and-tumour-tissue
#7
V Hrabal, M Nekulová, R Nenutil, J Holčaková, P J Coates, B Vojtěšek
BACKGROUND: PLA (proximity ligation assay) can be used for detection of protein-protein interactions in situ directly in cells and tissues. Due to its high sensitivity and specificity it is useful for detection, localization and quantification of protein complexes with single molecule resolution. One of the mechanisms of mutated p53 gain of function is formation of proten-protein complexes with other members of p53 family - p63 and p73. These interactions influences chemosensitivity and invasivity of cancer cells and this is why these complexes are potential targets of anti-cancer therapy...
2017: Klinická Onkologie: Casopis Ceské a Slovenské Onkologické Spolecnosti
https://www.readbyqxmd.com/read/28456688/native-metabotropic-glutamate-receptor-4-depresses-synaptic-transmission-through-an-unusual-g%C3%AE-q-transduction-pathway
#8
Solenne Chardonnet, Thomas Bessiron, Cathy Isaura Ramos, Raoudha Dammak, Marie-Ange Richard, Céline Boursier, Christelle Cadilhac, Frédéric M Coquelle, Simon Bossi, Fabrice Ango, Pierre Le Maréchal, Paulette Decottignies, Catherine Berrier, Heather McLean, Hervé Daniel
In cerebellar cortex, mGlu4 receptors located on parallel fibers play an essential role in normal motor function, but the molecular mechanisms involved are not yet completely understood. Using a strategy combining biochemical and electrophysiological approaches in the rodent cerebellum, we demonstrate that presynaptic mGlu4 receptors control synaptic transmission through an atypical activation of Gαq proteins. First, the Gαq subunit, PLC and PKC signaling proteins present in cerebellar extracts are retained on affinity chromatography columns grafted with different sequences of the cytoplasmic domain of mGlu4 receptor...
April 26, 2017: Neuropharmacology
https://www.readbyqxmd.com/read/28451223/imaging-specific-newly-synthesized-proteins-within-cells-by-fluorescence-resonance-energy-transfer
#9
Linfeng Sheng, Lesi Cai, Jie Liu, Sichun Zhang, Jing-Juan Xu, Xinrong Zhang, Hong-Yuan Chen
Metabolic azide amino acid labelling followed by the use of bioorthogonal chemistry is an efficient technique for imaging newly synthesized proteins. Recently, AHA-labelling together with the proximity-ligation assay was used to identify newly synthesized proteins of interest (POI) (Tom Dieck et al., Nat. Meth. 2015, 12, 411). Here we build on this study replacing the proximity-ligation assay with FRET to improve the spatial resolution. Herein, we develop a FRET-based strategy for imaging the newly synthesized endogenous POI within cells: a FRET acceptor is installed onto the newly synthesized proteins via click chemistry, and a FRET donor onto the POI via immunocytochemistry...
January 1, 2017: Chemical Science
https://www.readbyqxmd.com/read/28430879/role-of-phosphatase-and-tensin-homolog-pten-in-hypoxic-pulmonary-vasoconstriction
#10
Adrienn Krauszman, Tak Mak, Katalin Szaszi, Wolfgang M Kuebler
AIMS: Hypoxic pulmonary vasoconstriction (HPV) redistributes blood flow from poorly ventilated to better aerated areas in the lung, thereby optimizing ventilation-perfusion ratio (V/Q). Pulmonary artery smooth muscle cell (PASMC) contraction in response to hypoxia is triggered by Ca2+ influx via transient receptor potential canonical 6 (TRPC6) cation channels that have translocated to caveolae in the plasma membrane. Since phosphatase and tensin homolog (PTEN) was suggested to regulate TRPC6 in endothelial cells, we aimed to define its role in the hypoxic response of PASMCs and as a putative mediator of HPV...
April 18, 2017: Cardiovascular Research
https://www.readbyqxmd.com/read/28427827/antiviral-screen-identifies-ev71-inhibitors-and-reveals-camptothecin-target-dna-topoisomerase-1-as-a-novel-ev71-host-factor
#11
Kan Xing Wu, Justin Jang-Hann Chu
Enterovirus 71 (EV71) is one of the causative agents of hand, foot and mouth disease (HFMD) associated with severe neurological disease. EV71's pathogenesis remains poorly understood and the lack of approved antiviral has led to its emergence as a clinically important neurotropic virus. The goals of this study were to: (i) identify novel anti-EV71 compounds that may serve as lead molecules for therapeutics; and (ii) investigate their targets in downstream studies. We screened a 502-compound library of highly purified natural products for anti-EV71 activities in a cell-based immunofluorescence assay that were then confirmed in viral plaque reduction assays...
April 17, 2017: Antiviral Research
https://www.readbyqxmd.com/read/28424276/thrombin-induced-podocyte-injury-is-protease-activated-receptor-dependent
#12
Ruchika Sharma, Amanda P Waller, Shipra Agrawal, Katelyn J Wolfgang, Hiep Luu, Khurrum Shahzad, Berend Isermann, William E Smoyer, Marvin T Nieman, Bryce A Kerlin
Nephrotic syndrome is characterized by massive proteinuria and injury of specialized glomerular epithelial cells called podocytes. Studies have shown that, whereas low-concentration thrombin may be cytoprotective, higher thrombin concentrations may contribute to podocyte injury. We and others have demonstrated that ex vivo plasma thrombin generation is enhanced during nephrosis, suggesting that thrombin may contribute to nephrotic progression. Moreover, nonspecific thrombin inhibition has been shown to decrease proteinuria in nephrotic animal models...
April 19, 2017: Journal of the American Society of Nephrology: JASN
https://www.readbyqxmd.com/read/28415614/connexin40-controls-endothelial-activation-by-dampening-nfkb-activation
#13
Jean-Francois Denis, Ke Ludwig Scheckenbach, Anna Pfenniger, Merlijn J Meens, Rob Krams, Lucile Miquerol, Steven Taffet, Marc Chanson, Mario Delmar, Brenda R Kwak
Connexins are proteins forming gap junction channels for intercellular communication. Connexin40 (Cx40) is highly expressed by endothelial cells (ECs) of healthy arteries but this expression is lost in ECs overlying atherosclerotic plaques. Low/oscillatory shear stress observed in bends and bifurcations of arteries is atherogenic partly through activation of the pro-inflammatory NFκB pathway in ECs. In this study, we investigated the relation between shear stress, Cx40 and NFκB. Shear stress-modifying casts were placed around carotid arteries of mice expressing eGFP under the Cx40 promoter (Cx40+/eGFP)...
March 22, 2017: Oncotarget
https://www.readbyqxmd.com/read/28414308/complex-regulation-of-lcor-signaling-in-breast-cancer-cells
#14
S Jalaguier, C Teyssier, T Nait Achour, A Lucas, S Bonnet, C Rodriguez, N Elarouci, M Lapierre, V Cavaillès
Ligand-dependent corepressor (LCoR) is a transcriptional repressor of ligand-activated estrogen receptors (ERs) and other transcription factors that acts both by recruiting histone deacetylases and C-terminal binding proteins. Here, we first studied LCOR gene expression in breast cancer cell lines and tissues. We detected two mRNAs variants, LCoR and LCoR2 (which encodes a truncated LCoR protein). Their expression was highly correlated and localized in discrete nuclear foci. LCoR and LCoR2 strongly repressed transcription, inhibited estrogen-induced target gene expression and decreased breast cancer cell proliferation...
April 17, 2017: Oncogene
https://www.readbyqxmd.com/read/28393224/overexpression-of-p53-protein-is-a-marker-of-poor-prognosis-in-mexican-women-with-breast-cancer
#15
Jisela Dimas-González, Vilma Maldonado-Lagunas, José Díaz-Chávez, María Eugenia López-Arellano, José Muñoz-Camacho, Marco Antonio Terán-Porcayo, Alfredo Lagunas-Martínez
Breast cancer (BC) is a disease with different clinical, histological and molecular characteristics, frequently presenting mutated tumour-suppressing genes and oncogenes. P53 is a known tumour suppressor that is often mutated in BC; several mutations in p53 inhibit its role as a transcriptional repressor of several oncogenes. Topoisomerase 2α (TOP2α) is a gene target of p53, and it is also a known target for anthracyclines. The aim of the present study, was to analyse the genetic alterations of p53 and TOP2α genes and their levels of protein expression, as well as their association with survival in Mexican women with BC...
May 2017: Oncology Reports
https://www.readbyqxmd.com/read/28387307/stomatin-modulates-the-activity-of-the-anion-exchanger-1-ae1-slc4a1
#16
Sandrine Genetet, Alexandra Desrames, Youcef Chouali, Pierre Ripoche, Claude Lopez, Isabelle Mouro-Chanteloup
Anion Exchanger 1 (AE1) and stomatin are integral proteins of the red blood cell (RBC) membrane. Erythroid and kidney AE1 play a major role in HCO3(-) and Cl(-) exchange. Stomatins down-regulate the activity of many channels and transporters. Biochemical studies suggested an interaction of erythroid AE1 with stomatin. Moreover, we previously reported normal AE1 expression level in stomatin-deficient RBCs. Here, the ability of stomatin to modulate AE1-dependent Cl(-)/HCO3(-) exchange was evaluated using stopped-flow methods...
April 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28379402/slp-2-interacts-with-parkin-in-mitochondria-and-prevents-mitochondrial-dysfunction-in-parkin-deficient-human-ipsc-derived-neurons-and-drosophila
#17
Alessandra Zanon, Sreehari Kalvakuri, Aleksandar Rakovic, Luisa Foco, Marianna Guida, Christine Schwienbacher, Alice Serafin, Franziska Rudolph, Michaela Trilck, Anne Grünewald, Nancy Stanslowsky, Florian Wegner, Valentina Giorgio, Alexandros A Lavdas, Rolf Bodmer, Peter P Pramstaller, Christine Klein, Andrew A Hicks, Irene Pichler, Philip Seibler
Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins...
April 3, 2017: Human Molecular Genetics
https://www.readbyqxmd.com/read/28377570/flow-cytometric-measurement-of-blood-cells-with-bcr-abl1-fusion-protein-in-chronic-myeloid-leukemia
#18
Liza Löf, Linda Arngården, Ulla Olsson-Strömberg, Benjamin Siart, Mattias Jansson, Joakim S Dahlin, Ingrid Thörn, Lisa Christiansson, Monica Hermansson, Anders Larsson, Erik Ahlstrand, Göran Wålinder, Ola Söderberg, Richard Rosenquist, Ulf Landegren, Masood Kamali-Moghaddam
Chronic myeloid leukemia (CML) is characterized in the majority of cases by a t(9;22)(q34;q11) translocation, also called the Philadelphia chromosome, giving rise to the BCR-ABL1 fusion protein. Current treatment with tyrosine kinase inhibitors is directed against the constitutively active ABL1 domain of the fusion protein, and minimal residual disease (MRD) after therapy is monitored by real-time quantitative PCR (RQ-PCR) of the fusion transcript. Here, we describe a novel approach to detect and enumerate cells positive for the BCR-ABL1 fusion protein by combining the in situ proximity ligation assay with flow cytometry as readout (PLA-flow)...
April 4, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28376192/deciphering-the-role-of-oncogenic-mitfe318k-in-senescence-delay-and-melanoma-progression
#19
Caroline Bonet, Flavie Luciani, Jean-François Ottavi, Justine Leclerc, Fanélie-Marie Jouenne, Marina Boncompagni, Karine Bille, Véronique Hofman, Guillaume Bossis, Gian Marco de Donatis, Thomas Strub, Yann Cheli, Mickaël Ohanna, Frédéric Luciano, Sandrine Marchetti, Stéphane Rocchi, Marie-Christine Birling, Marie-Françoise Avril, Nicolas Poulalhon, Thomas Luc, Corine Bertolotto
Background: MITF encodes an oncogenic lineage-specific transcription factor in which a germline mutation ( MITFE318K ) was identified in human patients predisposed to both nevus formation and, among other tumor types, melanoma. The molecular mechanisms underlying the oncogenic activity of MITF E318K remained uncharacterized. Methods: Here, we compared the SUMOylation status of endogenous MITF by proximity ligation assay in melanocytes isolated from wild-type (n = 3) or E318K (n = 4) MITF donors...
August 1, 2017: Journal of the National Cancer Institute
https://www.readbyqxmd.com/read/28369339/hic-spector-a-matrix-library-for-spectral-and-reproducibility-analysis-of-hi-c-contact-maps
#20
Koon-Kiu Yan, Galip Gürkan Yardimci, Chengfei Yan, William S Noble, Mark Gerstein
Summary: Genome-wide proximity ligation based assays like Hi-C have opened a window to the 3D organization of the genome. In so doing, they present data structures that are different from conventional 1D signal tracks. To exploit the 2D nature of Hi-C contact maps, matrix techniques like spectral analysis are particularly useful. Here, we present HiC-spector, a collection of matrix-related functions for analyzing Hi-C contact maps. In particular, we introduce a novel reproducibility metric for quantifying the similarity between contact maps based on spectral decomposition...
March 22, 2017: Bioinformatics
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