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Pull down assay

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https://www.readbyqxmd.com/read/28930151/perk-signal-modulated-protein-translation-promotes-the-survivability-of-dengue-2-virus-infected-mosquito-cells-and-extends-viral-replication
#1
Jiun-Nan Hou, Tien-Huang Chen, Yi-Hsuan Chiang, Jing-Yun Peng, Tsong-Han Yang, Chih-Chieh Cheng, Eny Sofiyatun, Cheng-Hsun Chiu, Chuan Chiang-Ni, Wei-June Chen
Survival of mosquitoes from dengue virus (DENV) infection is a prerequisite of viral transmission to the host. This study aimed to see how mosquito cells can survive the infection during prosperous replication of the virus. In C6/36 cells, global protein translation was shut down after infection by DENV type 2 (DENV2). However, it returned to a normal level when infected cells were treated with an inhibitor of the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway. Based on a 7-Methylguanosine 5'-triphosphate (m7GTP) pull-down assay, the eukaryotic translation initiation factor 4F (eIF4F) complex was also identified in DENV2-infected cells...
September 20, 2017: Viruses
https://www.readbyqxmd.com/read/28918251/hmg-box-factor-soxd-sox15-and-homeodomain-containing-factor-xanf1-hesx1-directly-interact-and-regulate-the-expression-of-xanf1-hesx1-during-early-forebrain-development-in-xenopus-laevis
#2
Natalia Y Martynova, Fedor M Eroshkin, Eugeny E Оrlov, Andrey G Zaraisky
The homeodomain-containing transcription factor Anf (also known as Rpx/Hesx1 in mammals) plays an important role during the forebrain development in vertebrates. Here we demonstrate the ability of the Xenopus laevis Anf, Xanf1/Hesx1, to directly bind SRY-related HMG-box-containing transcription factor SoxD/Sox15 and to cooperate with the latter during regulating of the expression of Xanf1/Hesx1 own gene. As we have shown by GST pull-down, EMSA and the luciferase reporter assays, Xanf1/Hesx1 and SoxD/Sox15 bind the Xanf1/Hesx1 promoter region counteracting the inhibitory effect of Xanf1/Hesx1 alone...
September 13, 2017: Gene
https://www.readbyqxmd.com/read/28914468/the-toxic-alpha-gliadin-peptide-31-43-enters-cells-without-a-surface-membrane-receptor
#3
Paolella Gaetana, Lepretti Marilena, Martucciello Stefania, Nanayakkara Merlin, Auricchio Salvatore, Esposito Carla, Barone Maria Vittoria, Caputo Ivana
Alpha-gliadin peptide 31-43 is considered to be the main peptide responsible for the innate immune response in celiac disease patients. Recent evidence indicates that peptide 31-43 rapidly enters cells and interacts with the early endocytic vesicular compartment. However, the mechanism of its uptake is not completely understood. Our aim is to characterize, isolate and identify possible cell surface proteins involved in peptide 31-43 internalization by Caco-2 cells. In this study, we used a chemical cross-linker to block peptide 31-43 on cell surface proteins, and pulled-down peptide-proteins complexes using antibodies raised against peptide 31-43...
September 15, 2017: Cell Biology International
https://www.readbyqxmd.com/read/28902349/tug1-promotes-osteosarcoma-tumorigenesis-by-upregulating-ezh2-expression-via-mir-144-3p
#4
Jiaqing Cao, Xinyou Han, Xin Qi, Xiangyun Jin, Xiaolin Li
lncRNA-TUG1 (Taurine upregulated 1) is up-regulated and highly correlated with poor prognosis and disease status in osteosarcoma. TUG1 knockdown inhibits osteosarcoma cell proliferation, migration and invasion, and promotes apoptosis. However, its mechanism of action has not been well addressed. Growing evidence documented that lncRNA works as competing endogenous (ce)RNAs to modulate the expression and biological functions of miRNA. As a putative combining target of TUG1, miR-144-3p has been associated with the progress of osteosarcoma...
August 30, 2017: International Journal of Oncology
https://www.readbyqxmd.com/read/28902164/slbir3-negatively-regulates-pamp-responses-and-cell-death-in-tomato
#5
Shuhua Huang, Shuming Nie, Shufen Wang, Jianwei Liu, Yanfeng Zhang, Xiaofeng Wang
Bri1-associated kinase 1 (BAK1)-interacting receptor-like kinase (BIR) proteins have been shown to play important roles in regulating growth and development, pathogen associated molecular pattern (PAMP)-triggered immunity (PTI) responses, and cell death in the model plant, Arabidopsis thaliana. We identified four BIR family members in tomato (Solanum lycopersicum), including SlBIR3, an ortholog of AtBIR3 from A. thaliana. SlBIR3 is predicted to encode a membrane localized non-arginine-aspartate (non-RD) kinase that, based on protein sequence, does not have autophosphorylation activity but that can be phosphorylated in vivo...
September 13, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/28895884/interaction-of-recombinant-gallus-gallus-sept5-and-brain-proteins-of-h5n1-avian-influenza-virus-infected-chickens
#6
Jasmine Elanie Khairat, Vinod Balasubramaniam, Iekhsan Othman, Abdul Rahman Omar, Sharifah Syed Hassan
Septin forms a conserved family of cytoskeletal guanosine triphosphate (GTP) binding proteins that have diverse roles in protein scaffolding, vesicle trafficking, and cytokinesis. The involvement of septins in infectious viral disease pathogenesis has been demonstrated by the upregulation of SEPT5 protein and its mRNA in brain tissues of H5N1-infected chickens, thus, providing evidence for the potential importance of this protein in the pathogenesis of neurovirulence caused by the avian influenza virus. In this study, cloning, expression, and purification of Gallus gallus SEPT5 protein was performed in Escherichia coli...
September 12, 2017: Proteomes
https://www.readbyqxmd.com/read/28889305/detection-of-cyclic-di-gmp-binding-proteins-utilizing-a-biotinylated-cyclic-di-gmp-pull-down-assay
#7
Jacob R Chambers, Karin Sauer
Cyclic di-GMP is an important regulatory messenger molecule that often directly interacts with proteins to alter function. It is therefore important to find potential c-di-GMP binding proteins and verify a direct interaction between them. Here, we describe a pull-down assay using biotinylated-c-di-GMP to capture a specific protein of interest followed by immunoblot analysis to determine relative protein abundance. This method also allows for addition of both specific and nonspecific competitors to determine specificity of c-di-GMP-protein binding...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28889303/detection-of-c-di-gmp-responsive-dna-binding
#8
Jacob R Chambers, Karin Sauer
Modulation of signal transduction via binding of the secondary messenger molecule cyclic di-GMP to effector proteins is a near universal regulatory schema in bacteria. In particular, direct binding of c-di-GMP to transcriptional regulators has been shown to alter gene expression of a variety of processes. Here, we illustrate a pull-down-based DNA:protein binding reaction to determine the relative importance of c-di-GMP in the binding affinity of a target protein to specific DNA sequences. Specifically, the pull-down-based assay enables DNA binding to be analyzed with differing concentrations of c-di-GMP in the absence/presence of specific and nonspecific competitors...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28888848/prevention-of-oxytosis-induced-c-raf-down-regulation-by-arylthio-cyclopentenone-prostaglandins-is-neuroprotective
#9
Shoko Shibata, Kyoji Furuta, Kentaro Oh-Hashi, Hiroshi Ueda, Kazutoshi Kiuchi, Yoko Hirata
Prolonged exposure to high concentrations of glutamate leads to cell type specific glutathione depletion and resulting oxidative stress, known as oxytosis. As a result of glutathione depletion, accumulation of reactive oxygen species and Ca(2+) influx are increased; however, the specific target of oxytosis has yet to be identified. In the present study, we focused on the effect of glutamate-induced oxidative stress on the extracellular-regulated protein kinase (ERK) pathway using the murine hippocampal HT22 cell line...
September 6, 2017: Toxicology
https://www.readbyqxmd.com/read/28888655/the-matrix-protein-of-vesicular-stomatitis-virus-inhibits-host-directed-transcription-of-target-genes-via-interaction-with-the-tfiih-subunit-p8
#10
Wei Pan, Deguang Song, Wenqi He, Huijun Lu, Yungang Lan, Juanzhen Tong, Feng Gao, Kui Zhao
In response to viral infection, the host innate antiviral response is elicited to limit viral replication. Many viruses have evolved various strategies to circumvent the host antiviral response. It has been reported that matrix (M) protein of vesicular stomatitis virus (VSV) can inhibit host gene expression to evade the host innate immune response. However, the molecular mechanism remains unclear. Here, we demonstrated that VSV M protein inhibited transcription of a reporter gene transfected into BSR-T7/5 cells...
September 2017: Veterinary Microbiology
https://www.readbyqxmd.com/read/28887166/dusp14-protects-against-hepatic-ischemia-reperfusion-injury-via-tak1-suppression
#11
Xiaozhan Wang, Wenzhe Mao, Chun Fang, Song Tian, Xueyong Zhu, Ling Yang, Zan Huang, Hongliang Li
BACKGROUND AND AIMS: Hepatic ischemia-reperfusion (I/R) injury is characterized by severe inflammation and extensive cell death. Multiple signaling pathways, including NF-κB and MAPK/Jnk, play important roles in this process. Identifying the unknown critical regulators of these signaling pathways may provide potential targets for therapeutic application. Dual specific phosphatase 14 (DUSP14) acts as a negative regulator of NF-κB signaling. However, the function of Dusp14 in hepatic I/R injury remains unknown...
September 5, 2017: Journal of Hepatology
https://www.readbyqxmd.com/read/28881612/the-b-box-module-of-cyld-is-responsible-for-its-intermolecular-interaction-and-cytoplasmic-localization
#12
Songbo Xie, Miao Chen, Siqi Gao, Tao Zhong, Peng Zhou, Dengwen Li, Jun Zhou, Jinmin Gao, Min Liu
The tumor suppressor protein cylindromatosis (CYLD), as a microtubule-associated deubiquitinase, plays a pivotal role in a wide range of cellular activities, including innate immunity, cell division, and ciliogenesis. Structural characterization reveals a small zinc-binding B-box inserted within the ubiquitin specific protease (USP) domain of CYLD; however, the exact role for this module remains yet to be elucidated. Here we identify a critical role for the B-box in facilitating the intermolecular interaction and subcellular localization of CYLD...
August 1, 2017: Oncotarget
https://www.readbyqxmd.com/read/28876241/structural-insights-into-the-interaction-of-the-conserved-mammalian-proteins-gapr-1-and-beclin-1-a-key-autophagy-protein
#13
Yue Li, Yuting Zhao, Minfei Su, Karen Glover, Srinivas Chakravarthy, Christopher L Colbert, Beth Levine, Sangita C Sinha
Mammalian Golgi-associated plant pathogenesis-related protein 1 (GAPR-1) is a negative autophagy regulator that binds Beclin 1, a key component of the autophagosome nucleation complex. Beclin 1 residues 267-284 are required for binding GAPR-1. Here, sequence analyses, structural modeling, mutagenesis combined with pull-down assays, X-ray crystal structure determination and small-angle X-ray scattering were used to investigate the Beclin 1-GAPR-1 interaction. Five conserved residues line an equatorial GAPR-1 surface groove that is large enough to bind a peptide...
September 1, 2017: Acta Crystallographica. Section D, Structural Biology
https://www.readbyqxmd.com/read/28868744/a-putative-spermidine-synthase-interacts-with-flagellar-switch-protein-flim-and-regulates-motility-in-helicobacter-pylori
#14
Huawei Zhang, Kwok Ho Lam, Wendy Wai Ling Lam, Sandra Yuen Yuen Wong, Vera Sau Fong Chan, Shannon Wing Ngor Au
The flagellar motor is an important virulence factor in infection by many bacterial pathogens. Motor function can be modulated by chemotactic proteins and recently-appreciated proteins that are not part of the flagellar or chemotaxis systems. How these latter proteins affect flagellar activity is not fully understood. Here, we identified spermidine synthase SpeE as an interacting partner of switch protein FliM in Helicobacter pylori using pull-down assay and mass spectrometry. To understand how SpeE contributes to flagellar motility, a speE-null mutant was generated and its motility behavior was evaluated...
September 4, 2017: Molecular Microbiology
https://www.readbyqxmd.com/read/28868548/developing-antineoplastic-agents-that-target-peroxisomal-enzymes-cytisine-linked-isoflavonoids-as-inhibitors-of-hydroxysteroid-17-beta-dehydrogenase-4-hsd17b4
#15
Mykhaylo S Frasinyuk, Wen Zhang, Przemyslaw Wyrebek, Tianxin Yu, Xuehe Xu, Vitaliy M Sviripa, Svitlana P Bondarenko, Yanqi Xie, Huy X Ngo, Andrew J Morris, James L Mohler, Michael V Fiandalo, David S Watt, Chunming Liu
Cytisine-linked isoflavonoids (CLIFs) inhibited PC-3 prostate and LS174T colon cancer cell proliferation by inhibiting a peroxisomal bifunctional enzyme. A pull-down assay using a biologically active, biotin-modified CLIF identified the target of these agents as the bifunctional peroxisomal enzyme, hydroxysteroid 17β-dehydrogenase-4 (HSD17B4). Additional studies with truncated versions of HSD17B4 established that CLIFs specifically bind the C-terminus of HSD17B4 and selectively inhibited the enoyl CoA hydratase but not the d-3-hydroxyacyl CoA dehydrogenase activity...
September 20, 2017: Organic & Biomolecular Chemistry
https://www.readbyqxmd.com/read/28864835/neat1-accelerates-the-progression-of-liver-fibrosis-via-regulation-of-microrna-122-and-kruppel-like-factor-6
#16
Fujun Yu, Zhe Jiang, Bicheng Chen, Peihong Dong, Jianjian Zheng
Long non-coding RNAs (lncRNAs) have been reported to be involved in many important biological processes including proliferation, apoptosis, differentiation, and survival. Recently, nuclear paraspeckle assembly transcript 1 (NEAT1), a novel lncRNA, serves as a crucial regulator in tumors. However, the biological role of NEAT1 in liver fibrosis is largely unknown. In this study, the role of NEAT1 was explored in primary mouse hepatic stellate cells (HSCs) and carbon tetrachloride (CCl4)-induced mouse liver fibrosis models...
September 1, 2017: Journal of Molecular Medicine: Official Organ of the "Gesellschaft Deutscher Naturforscher und Ärzte"
https://www.readbyqxmd.com/read/28862774/deinococcus-radiodurans-dr1088-is-a-novel-recf-interacting-protein-that-stimulates-single-stranded-dna-annealing
#17
Kaiying Cheng, Guangzhi Xu, Hong Xu, Ye Zhao, Yuejin Hua
RecF, together with the recombination mediators RecO and RecR, is required in the RecFOR homologous recombination repair pathway in bacteria. In this study, a recF-dr1088 operon, which is highly conserved in the Deinococcus-Thermus phylum, was identified in Deinococcus radiodurans. Interaction between DRRecF and DR1088 was confirmed by yeast two-hybrid and pull-down assays. DR1088 exhibited some RecO-like biochemical properties including single/double-stranded DNA binding activity, ssDNA binding protein (SSB) replacement ability and ssDNA (with or without SSB) annealing activity...
September 1, 2017: Molecular Microbiology
https://www.readbyqxmd.com/read/28860123/circular-non-coding-rna-hipk3-mediates-retinal-vascular-dysfunction-in-diabetes-mellitus
#18
Kun Shan, Chang Liu, Bai-Hui Liu, Xue Chen, Rui Dong, Xin Liu, Yang-Yang Zhang, Ban Liu, Shu-Jie Zhang, Jia-Jian Wang, Sheng-Hai Zhang, Ji-Hong Wu, Chen Zhao, Biao Yan
Background -The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circRNA in retinal vascular dysfunction induced by diabetes. Methods -Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circHIPK3 expression pattern upon diabetes mellitus-related stresses...
August 31, 2017: Circulation
https://www.readbyqxmd.com/read/28855513/noninvasive-imaging-of-ras-activity-by-monomolecular-biosensor-based-on-split-luciferase-complementary-assay
#19
Liang Chen, Wei Bing Leng, De Zhi Li, Hong Wei Xia, Min Ren, Qiu Lin Tang, Qi Yong Gong, Fa Bao Gao, Feng Bi
Deregulated activity of Ras GTPases has been observed in many types of human cancers, and contributes to the diverse aspects of carcinogenesis. Although the significance in tumorigenesis has been widely accepted and many therapeutic drugs are under development, little attention has been dedicated to the development of sensors for the Ras activity in vivo. Therefore, based on the split firefly luciferase complementation strategy, we developed a monomolecular bioluminescent biosensor to image endogenous Ras activity in living subject...
August 30, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28855187/inhibition-of-smooth-muscle-contraction-and-arf-6-activity-by-the-inhibitor-for-cytohesin-gefs-secinh3-in-the-human-prostate
#20
Annika Herlemann, Patrick Keller, Melanie Schott, Alexander Tamalunas, Anna Ciotkowska, Beata Rutz, Yiming Wang, Qingfeng Yu, Raphaela Waidelich, Frank Strittmatter, Christian G Stief, Christian Gratzke, Martin Hennenberg
Prostate smooth muscle contraction is critical for etiology and treatment of male lower urinary tract symptoms (LUTS), and is promoted by small monomeric GTPases (RhoA, Rac). GTPases may be activated by guanosine nucleotide exchange factors (GEFs). GEFs of the cytohesin family may indirectly activate Rac, or ADP ribosolyation factor (ARF) GTPases directly. Here we investigated the expression of cytohesin family GEFs, and effects of the cytohesin inhibitor secinH3 on smooth muscle contraction and GTPase activities in human prostate tissues...
August 30, 2017: American Journal of Physiology. Renal Physiology
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