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Proximity ligation assay

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https://www.readbyqxmd.com/read/29330884/synapsin-iii-is-a-key-component-of-%C3%AE-synuclein-fibrils-in-lewy-bodies-of-pd-brains
#1
Francesca Longhena, Gaia Faustini, Tatiana Varanita, Michela Zaltieri, Vanessa Porrini, Isabella Tessari, Pietro Luigi Poliani, Cristina Missale, Barbara Borroni, Alessandro Padovani, Luigi Bubacco, Marina Pizzi, PierFranco Spano, Arianna Bellucci
Lewy bodies (LB) and Lewy neurites (LN), which are primarily composed of α-synuclein (α-syn), are neuropathological hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). We recently found that the neuronal phosphoprotein synapsin III (syn III) controls dopamine release via cooperation with α-syn and modulates α-syn aggregation. Here, we observed that LB and LN, in the substantia nigra of PD patients and hippocampus of one subject with DLB, displayed a marked immunopositivity for syn III...
January 13, 2018: Brain Pathology
https://www.readbyqxmd.com/read/29329781/dopamine-transporter-trafficking-is-regulated-by-neutral-sphingomyelinase-2-ceramide-kinase
#2
Jong Hoon Won, Seok Kyun Kim, In Chul Shin, Hae Chan Ha, Ji Min Jang, Moon Jung Back, Dae Kyong Kim
Dopamine (DA) reuptake is the primary mechanism to terminate dopaminergic transmission in the synaptic cleft. The dopamine transporter (DAT) has an important role in the regulation of DA reuptake. This study provides anatomical and physiological evidence that DAT recycling is regulated by ceramide kinase via the sphingomyelin pathway. First, the results show that DAT and neutral sphingomyelinase 2 (nSMase2) were successfully co-precipitated from striatal samples and were colocalized in the mouse striatum or PC12 cells...
January 9, 2018: Cellular Signalling
https://www.readbyqxmd.com/read/29321326/the-amino-terminus-of-hsv-1-glycoprotein-k-gk-is-required-for-gb-binding-to-akt-release-of-intracellular-calcium-and-fusion-of-the-viral-envelope-with-plasma-membranes
#3
Farhana Musarrat, Nithya Jambunathan, Paul J F Rider, V N Chouljenko, K G Kousoulas
Previously, we have shown that the amino terminus of glycoprotein K (gK) binds to the amino terminus of gB and that deletion of the amino terminal 38 amino acids of gK prevents virus infection of mouse trigeminal ganglia after ocular infection and virus entry into neuronal axons. Recently, it has been shown that gB binds to Akt during virus entry and induces Akt phosphorylation and intracellular calcium release. Proximity ligation and two-way immunoprecipitation assays using monoclonal antibodies against gB and Akt-1 (phospho S473) confirmed that HSV-1(McKrae) gB interacted with Akt-1 (phospo S473) during virus entry into human neuroblastoma (SK-N-SH) cells and induced release of intracellular calcium...
January 10, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29321137/novel-function-of-ceramide-for-regulation-of-mitochondrial-atp-release-in-astrocytes
#4
Ji-Na Kong, Zhihui Zhu, Yutaka Itokazu, Guanghu Wang, Michael B Dinkins, Liansheng Zhong, Hsuan-Pei Lin, Ahmed Elsherbini, Silvia Leanhart, Xue Jiang, Haiyan Qin, Wenbo Zhi, Stefka D Spassieva, Erhard Bieberich
We reported that amyloid beta peptide (Abeta42) activated neutral sphingomyelinase 2 (nSMase2) thereby increasing the concentration of the sphingolipid ceramide in astrocytes. Here, we show that Abeta42 induced mitochondrial fragmentation in wild type astrocytes, but not in nSMase2-deficient cells or astrocytes treated with Fumonisin B1 (FB1), an inhibitor of ceramide synthases. Unexpectedly, ceramide depletion was concurrent with rapid movements of mitochondria, indicating an unknown function of ceramide for mitochondria...
January 10, 2018: Journal of Lipid Research
https://www.readbyqxmd.com/read/29282288/structure-based-virtual-screening-identifies-small-molecule-inhibitor-of-the-profilin1-actin-interaction
#5
David Gau, Taber Lewis, Lee Mcdermott, Peter Wipf, David Koes, Partha Roy
Profilin1 (Pfn1) is an important regulator of the actin cytoskeleton and plays a vital role in many actin-based cellular processes. Therefore, identification of a small-molecule intervention strategy targeted against the Pfn1:actin interaction could have broad utility in cytoskeletal research and further our understanding of the role of Pfn1 in actin-mediated biological processes. Based on an already resolved Pfn1:actin complex crystal structure, we performed structure-based virtual screening of small molecule libraries to seek inhibitors of the Pfn1:actin interaction...
December 27, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29247325/tgf-%C3%AE-signaling-inhibits-canonical-bmp-signaling-pathway-during-palate-development
#6
Guohua Yuan, Yunyan Zhan, Xiaohui Gou, Yiping Chen, Guobin Yang
During early palate development, gene expression and regulation exhibit heterogeneity along the anterior-posterior axis. Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways play essential roles in secondary palatal formation but the exact relationship between the TGF-β and BMP pathways in palate development remains unknown. Here, we demonstrate that, during early secondary palate development, phospho-(p)Smad1/5/8 is highly expressed in the anterior palate but relatively lowly expressed in the posterior palate...
December 16, 2017: Cell and Tissue Research
https://www.readbyqxmd.com/read/29236046/advances-in-proteomic-techniques-for-cytokine-analysis-focus-on-melanoma-research
#7
REVIEW
Helena Kupcova Skalnikova, Jana Cizkova, Jakub Cervenka, Petr Vodicka
Melanoma is a skin cancer with permanently increasing incidence and resistance to therapies in advanced stages. Reports of spontaneous regression and tumour infiltration with T-lymphocytes makes melanoma candidate for immunotherapies. Cytokines are key factors regulating immune response and intercellular communication in tumour microenvironment. Cytokines may be used in therapy of melanoma to modulate immune response. Cytokines also possess diagnostic and prognostic potential and cytokine production may reflect effects of immunotherapies...
December 13, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29218528/proximity-ligation-assay-to-study-the-glut4-membrane-trafficking-machinery
#8
Dimitrios Kioumourtzoglou, Gwyn W Gould, Nia J Bryant
In this chapter a detailed protocol of proximity ligation assay (PLA) is described thoroughly. PLA is a technique that allows detection of protein associations in situ, providing a sensitive and selective approach for protein-protein interaction studies. We demonstrate the technique by applying it for trafficking studies of the facilitative glucose transporter GLUT4. Trafficking of GLUT4 from perinuclear depots to the plasma membrane is regulated by insulin in adipocytes and muscle cells, and mediated by formation of functional SNARE complexes containing Syntaxin4, SNAP23, and VAMP2...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29217667/critical-contribution-of-na-ca2-exchanger-to-the-ca2-mediated-vasodilation-activated-in-endothelial-cells-of-resistance-arteries
#9
Mauricio A Lillo, Pablo S Gaete, Mariela Puebla, Nicolás M Ardiles, Inés Poblete, Alvaro Becerra, Felipe Simon, Xavier F Figueroa
Na+-Ca2+ exchanger (NCX) contributes to control the intracellular free Ca2+ concentration ([Ca2+]i), but the functional activation of NCX reverse mode (NCXrm) in endothelial cells is controversial. We evaluated the participation of NCXrm-mediated Ca2+ uptake in the endothelium-dependent vasodilation of rat isolated mesenteric arterial beds. In phenylephrine-contracted mesenteries, the acetylcholine (ACh)-induced vasodilation was abolished by treatment with the NCXrm blockers SEA0400, KB-R7943, or SN-6. Consistent with that, the ACh-induced hyperpolarization observed in primary cultures of mesenteric endothelial cells and in smooth muscle of isolated mesenteric resistance arteries was attenuated by KB-R7943 and SEA0400, respectively...
December 7, 2017: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/29205397/effects-of-long-term-alcohol-drinking-on-the-dopamine-d2-receptor-gene-expression-and-heteroreceptor-complexes-in-the-striatum-in-rats
#10
Kristin Feltmann, Dasiel Oscar Borroto-Escuela, Joëlle Rüegg, Luca Pinton, Thatiane de Oliveira Sergio, Manuel Narváez, Antonio Jimenez-Beristain, Tomas J Ekström, Kjell Fuxe, Pia Steensland
BACKGROUND: Reduced dopamine D2 receptor (D2R) ligand binding has repeatedly been demonstrated in the striatum of humans with alcohol use disorder (AUD). The attenuated D2R binding has been suggested to reflect a reduced D2R density, which in turn has been proposed to drive craving and relapse. However, results from rodent studies addressing the effects of alcohol drinking on D2R density have been inconsistent. METHODS: A validated alcohol drinking model (intermittent access to 20% alcohol) in Wistar rats was used to study the effects of voluntary alcohol drinking (at least 12 weeks) on the D2R in the striatum compared to age-matched alcohol-naïve control rats...
December 5, 2017: Alcoholism, Clinical and Experimental Research
https://www.readbyqxmd.com/read/29199076/tension-dependent-stretching-activates-zo-1-to-control-the-junctional-localization-of-its-interactors
#11
Domenica Spadaro, Shimin Le, Thierry Laroche, Isabelle Mean, Lionel Jond, Jie Yan, Sandra Citi
Tensile forces regulate epithelial homeostasis, but the molecular mechanisms behind this regulation are poorly understood. Using structured illumination microscopy and proximity ligation assays, we show that the tight junction protein ZO-1 exists in stretched and folded conformations within epithelial cells, depending on actomyosin-generated force. We also show that ZO-1 and ZO-2 regulate the localization of the transcription factor DbpA and the tight junction membrane protein occludin in a manner that depends on the organization of the actin cytoskeleton, myosin-II activity, and substrate stiffness, resulting in modulation of gene expression, cell proliferation, barrier function, and cyst morphogenesis...
November 30, 2017: Current Biology: CB
https://www.readbyqxmd.com/read/29190807/identification-of-blcap-as-a-novel-stat3-interaction-partner-in-bladder-cancer
#12
Irina Gromova, Sofia Svensson, Pavel Gromov, José M A Moreira
Bladder cancer associated protein (Blcap) expression is commonly down-regulated in invasive bladder cancer, and may have prognostic value given that its expression is negatively correlated with patient survival. We have previously investigated the expression patterns and cellular localization of Blcap in bladder cancer, where we found that about 20% of the lesions examined displayed strong nuclear expression of Blcap, and that this phenotype was associated with overall poor disease outcome. Here we report on the analysis of possible functional associations between nuclear expression of Blcap and canonical signaling pathways...
2017: PloS One
https://www.readbyqxmd.com/read/29187380/the-beach-domain-containing-protein-nbeal2-interacts-with-dock7-sec16a-and-vac14
#13
Louisa Mayer, Maria Jasztal, Mercedes Pardo, Salvadora Aguera de Haro, Janine Collins, Tadbir K Bariana, Peter A Smethurst, Luigi Grassi, Romina Petersen, Paquita Nurden, Rémi Favier, Lu Yu, Stuart Meacham, William J Astle, Jyoti Choudhary, Wyatt W Yue, Willem H Ouwehand, Jose A Guerrero
Mutations in NBEAL2, the gene encoding the scaffolding protein Nbeal2 are causal of Gray Platelet Syndrome (GPS), a rare recessive bleeding disorder characterized by platelets lacking α-granules and progressive marrow fibrosis. We present here the interactome of Nbeal2 with additional validation by reverse immunoprecipitation of Dock7, Sec16a and Vac14 as interactors of Nbeal2. We show that GPS-causing mutations in its BEACH domain have profound and possible effects on the interaction with Dock7 and Vac14, respectively...
November 29, 2017: Blood
https://www.readbyqxmd.com/read/29184507/kir2-1-nav1-5-channel-complexes-are-differently-regulated-than-kir2-1-and-nav1-5-channels-alone
#14
Raquel G Utrilla, Paloma Nieto-Marín, Silvia Alfayate, David Tinaquero, Marcos Matamoros, Marta Pérez-Hernández, Sandra Sacristán, Lorena Ondo, Raquel de Andrés, F Javier Díez-Guerra, Juan Tamargo, Eva Delpón, Ricardo Caballero
Cardiac Kir2.1 and Nav1.5 channels generate the inward rectifier K+ (IK1) and the Na+ (INa) currents, respectively. There is a mutual interplay between the ventricular INa and IK1 densities, because Nav1.5 and Kir2.1 channels exhibit positive reciprocal modulation. Here we compared some of the biological properties of Nav1.5 and Kir2.1 channels when they are expressed together or separately to get further insights regarding their putative interaction. First we demonstrated by proximity ligation assays (PLAs) that in the membrane of ventricular myocytes Nav1...
2017: Frontiers in Physiology
https://www.readbyqxmd.com/read/29180608/t-darpp-activates-igf-1r-signaling-to-regulate-glucose-metabolism-in-trastuzumab-resistant-breast-cancer-cells
#15
Gal Lenz, Angelica Hamilton, Shuhui Geng, Teresa Hong, Markus Kalkum, Jamil Momand, Susan E Kane, Janice Huss
PURPOSE: Increased glycolysis and glucose dependence is a hallmark of malignancy that enables tumors to maximize cell proliferation. In HER2+ cancers, an increase in glycolytic capacity is associated with trastuzumab resistance. IGF-1R activation and t-Darpp over-expression both confer trastuzumab resistance in breast cancer. We therefore investigated a role for IGF-1R and t-Darpp in regulating glycolytic capacity in HER2+ breast cancers. EXPERIMENTAL DESIGN: We examined the relationship between t-Darpp and IGF-1R expression in breast tumors and their respective relationships with patient survival...
November 27, 2017: Clinical Cancer Research: An Official Journal of the American Association for Cancer Research
https://www.readbyqxmd.com/read/29170752/proximal-ligation-assay-pla-on-lung-tissue-and-cultured-macrophages-to-demonstrate-protein-protein-interaction
#16
Roberto Mendez, Santanu Banerjee
In this protocol, we describe proximal ligation assay (PLA), an antibody-based detection method for protein-protein interaction. This method relies on specific binding of individual primary antibodies to the two putative interacting proteins. The primary antibodies need to have different hosts. The secondary antibodies against the two hosts have complementary oligonucleotide moieties attached to them. If the two antigens are in close proximity (presumably interacting with each other), the complementary oligonucleotides can anneal and fluorescent nucleotides can be incorporated in a single DNA polymerization step...
November 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/29170129/rbfox-family-proteins-make-the-homo-and-hetero-oligomeric-complexes
#17
Sunkyung Choi, Yong-Eun Kim, Jae Whan Kim, Namjoon Cho, Seonghye Cheon, Kee K Kim
Rbfox family of proteins that consists of Rbfox1, Rbfox2, and Rbfox3 in mammals regulates alternative pre-mRNA splicing in various tissues via direct binding to their RNA binding element. Although many studies have indicated the splicing activity of each member of the Rbfox family, the interactions of Rbfox family proteins are largely unknown. Here, we have investigated interactions among Rbfox family proteins. Co-immunoprecipitation (Co-IP) and GST-pull down assays confirmed that Rbfox proteins form homo and hetero complexes...
November 20, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29169465/research-techniques-made-simple-emerging-methods-to-elucidate-protein-interactions-through-spatial-proximity
#18
Yonglu Che, Paul A Khavari
Interactions between proteins are essential for fundamental cellular processes, and the diversity of such interactions enables the vast variety of functions essential for life. A persistent goal in biological research is to develop assays that can faithfully capture different types of protein interactions to allow their study. A major step forward in this direction came with a family of methods that delineates spatial proximity of proteins as an indirect measure of protein-protein interaction. A variety of enzyme- and DNA ligation-based methods measure protein co-localization in space, capturing novel interactions that were previously too transient or low affinity to be identified...
December 2017: Journal of Investigative Dermatology
https://www.readbyqxmd.com/read/29164763/tmem207-hinders-the-tumour-suppressor-function-of-wwox-in-oral-squamous-cell-carcinoma
#19
Katsuaki Bunai, Hiroshi Okubo, Kimika Hano, Keisuke Inoue, Yusuke Kito, Chiemi Saigo, Toshiyuki Shibata, Tamotsu Takeuchi
The WW domain-containing oxidoreductase (WWOX) functions as a tumour suppressor in oral carcinogenesis. As aberrant TMEM207 expression may lead to tumour progression by hampering the tumour suppressor function of WWOX in various cancers, we explored the expression and pathobiological properties of TMEM207, focusing on the WWOX-mediated regulation of the HIF-1α pathway in oral squamous cell carcinoma (OSCC). TMEM207 immunoreactivity was detected in 40 of 90 OSCC samples but not in neighbouring non-tumorous epithelial tissues...
November 22, 2017: Journal of Cellular and Molecular Medicine
https://www.readbyqxmd.com/read/29157747/multiplex-proximity-ligation-assay-to-identify-potential-prognostic-biomarkers-for-improved-survival-in-locally-advanced-pancreatic-cancer-patients-treated-with-stereotactic-body-radiation-therapy
#20
Avani D Rao, Yufei Liu, Rie von Eyben, Charles C Hsu, Chen Hu, Lauren M Rosati, Arti Parekh, Kendall Ng, Amy Hacker-Prietz, Lei Zheng, Timothy M Pawlik, Daniel A Laheru, Elizabeth M Jaffee, Matthew J Weiss, Dung T Le, Ralph H Hruban, Ana De Jesus-Acosta, Christopher L Wolfgang, Amol K Narang, Daniel T Chang, Albert C Koong, Joseph M Herman
PURPOSE: To explore seromarker levels for associations with outcomes in locally advanced pancreatic cancer (LAPC) patients who received chemotherapy and stereotactic body radiation therapy (SBRT). METHODS AND MATERIALS: Serum from LAPC patients in 2 prospective trials of hypofractionated SBRT (5-6.6 Gy × 5) was collected before SBRT. Proximity ligation assay quantified the expression levels of 36 pancreatic cancer-specific candidate seromarkers: Axl, BMP2, CA 125, CA 19-9, CEA, CXCL-1/6/9/10, EGFR, Gas6, Her2, IGF-2, IGFBP-2/3/7, IL-6/6Ra/7/8/12, mesothelin, MMP-1/2/3/7, osteopontin, PDGFRa, PDK1, PF4, RegIV, SPARC, TGF-β, VEGF-A/D, and YKL40...
October 12, 2017: International Journal of Radiation Oncology, Biology, Physics
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