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Pull down assay

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https://www.readbyqxmd.com/read/28344045/ezh2-mediated-%C3%AE-actin-methylation-needs-lncrna-tug1-and-promotes-the-cortex-cytoskeleton-formation-in-vsmcs
#1
Rong Chen, Peng Kong, Fan Zhang, Ya-Nan Shu, Xi Nie, Li-Hua Dong, Yan-Ling Lin, Xiao-Li Xie, Li-Li Zhao, Xiang-Jian Zhang, Mei Han
Recent studies have revealed that long non-coding RNAs (lncRNAs) participate in vascular homeostasis and pathophysiological conditions development. But still very few literatures elucidate the regulatory mechanism of non-coding RNAs in this biological process. Here we identified lncRNA taurine up-regulated gene 1 (TUG1) in rat vascular smooth muscle cells (VSMCs), and got 4612bp nucleotide sequence. The expression level of TUG1 RNA was increased in synthetic VSMCs by real-time PCR analysis. Meanwhile, the expression of enhancer of zeste homolog 2 (EZH2) (TUG1 binding protein) increased in cytoplasm of VSMCs under the same conditions...
March 23, 2017: Gene
https://www.readbyqxmd.com/read/28343945/as-human-lung-microvascular-endothelia-achieve-confluence-src-family-kinases-are-activated-and-tyrosine-phosphorylated-p120-catenin-physically-couples-neu1-sialidase-to-cd31
#2
Sang W Hyun, Anguo Liu, Zhenguo Liu, Erik P Lillehoj, Joseph A Madri, Albert B Reynolds, Simeon E Goldblum
In postconfluent human pulmonary microvascular endothelial cell (HPMEC)s, NEU1 sialidase associates with and desialylates the src family kinase (SFK) substrate, CD31, and disrupts angiogenesis. We asked whether the NEU1-CD31 interaction might be SFK-driven. We found that normalized phospho-SFK (PY416) signal is increased in postconfluent HPMECs compared to subconfluent cells and prior SFK inhibition with PP2 or SU6656 completely blocked NEU1 association with and desialylation of CD31. Prior silencing of each of the four SFKs expressed in HPMECs, as well as CD31, dramatically reduced confluence-induced SFK activation...
March 24, 2017: Cellular Signalling
https://www.readbyqxmd.com/read/28343069/linc00152-promotes-cancer-progression-in-hepatitis-b-virus-associated-hepatocellular-carcinoma
#3
Xin Deng, Xiao Fang Zhao, Xing Qiu Liang, Ran Chen, Yi Feng Pan, Jian Liang
BACKGROUND: The X protein (HBx) plays as a key role in hepatocarcinogenesis associated with hepatitis B virus (HBV) infections. The study aimed to figure out the role of Linc00152 in hepatocellular carcinoma (HCC) and the association between the expression levels of Linc00152 and HBx. METHODS: QRT-PCR assays were applied to analyzed the expression levels of Linc00152 and HBx. Kaplan-Meier survival curve was performed to identify the association between LINC00152 and the over survival time (OS) in HCC patients...
March 23, 2017: Biomedicine & Pharmacotherapy, Biomédecine & Pharmacothérapie
https://www.readbyqxmd.com/read/28341660/the-noncoding-rna-linc-adamts5-cooperates-with-rreb1-to-protect-from-intervertebral-disc-degeneration-through-inhibiting-adamts5-expression
#4
Kun Wang, Yu Song, Wei Liu, Xinghuo Wu, Yukun Zhang, Shuai Li, Liang Kang, Ji Tu, Kangcheng Zhao, Wenbin Hua, Cao Yang
Previous studies have indicated the important roles of ADAMTS5 in intervertebral disc degeneration. However, the mechanisms that regulate ADAMTS5 expression in nuclear pulposus (NP) cells remain largely unknown. Evidence suggests that intergenic transcription may be associated with genes that encode transcriptional regulators. Here, we identified a long intergenic noncoding RNA, linc-ADAMTS5, that was transcribed in the opposite direction of ADAMTS5. In this study, through mining computational algorithm programs, and public available data sets, we identified Ras responsive element binding protein 1 (RREB1) as a crucial transcription factor regulating the expression of ADAMTS5 in NP cells...
March 24, 2017: Clinical Science (1979-)
https://www.readbyqxmd.com/read/28341602/cytosolic-yb-1-and-nsun2-are-the-only-proteins-recognizing-specific-motifs-present-in-mrnas-enriched-in-exosomes
#5
Olga A Kossinova, Alexander V Gopanenko, Svetlana N Tamkovich, Olga A Krasheninina, Alexey E Tupikin, Elena Kiseleva, Darya D Yanshina, Alexey A Malygin, Alia G Ven'yaminova, Marsel R Kabilov, Galina G Karpova
Exosomes, membranous vesicles secreted by various cells, are involved in intercellular communication and carry vast repertoires of RNAs and proteins. Processes mediating RNA sorting into exosomes are currently poorly understood. Using bioinformatics approaches, three structural motifs ACCAGCCU, CAGUGAGC and UAAUCCCA have been discovered as enriched in exosomal mRNAs and long noncoding RNAs. Here, utilizing short RNA hairpins, each containing one of the motifs, in a pull-down assay of cytosolic extract of human embryonic kidney 293 (HEK293) cells, we prove that multifunctional RNA-binding protein YB-1 specifically interacts with all three motifs, whereas methyltransferase NSUN2 recognizes only the motif CAGUGAGC...
March 21, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28337962/linc00052-promotes-gastric-cancer-cell-proliferation-and-metastasis-via-activating-wnt-%C3%AE-catenin-signaling-pathway
#6
Yuqiang Shan, Rongchao Ying, Zhong Jia, Wencheng Kong, Yi Wu, Sixin Zheng, Huicheng Jin
Gastric cancer (GC) is one of the most common malignant tumors of digestive system. The etiology of GC is complex and much more attention should be paid on the genetic factors. In this study, we explored the role and function of LINC00052 in GC. We applied qRT-PCR and northern blot to detect the expression of LINC00052 and found it was highly expressed during GC. We also investigated the effects of LINC00052 on tumor prognosis and progression, and found LINC00052 indicated poor prognosis and tumor progression...
March 23, 2017: Oncology Research
https://www.readbyqxmd.com/read/28336771/dimerization-in-lbd16-and-lbd18-transcription-factors-is-critical-for-lateral-root-formation
#7
Han Woo Lee, Na Young Kang, Shashank Pandey, Chuloh Cho, Sung Haeng Lee, Jungmook Kim
LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD)/ASYMMETRIC LEAVES2-LIKE (ASL)s (hereafter referred to as LBD) are plant-specific transcription factors that play important roles in a plethora of plant growth and development. The leucine-zipper-like coiled-coil motif in the LOB domain of the class I LBD proteins has been proposed to mediate protein dimerization, but it has not been experimentally assessed yet. LBD16 and LBD18 have been well characterized to play important roles in lateral root development in Arabidopsis...
March 23, 2017: Plant Physiology
https://www.readbyqxmd.com/read/28325828/the-prolyl-isomerase-pin1-is-a-novel-target-of-6-7-4-trihydroxyisoflavone-for-suppressing-esophageal-cancer-growth
#8
Tae Gyu Lim, Sung-Young Lee, Zhaoheng Duan, Mee-Hyun Lee, Hanyong Chen, Fangfang Liu, Kangdong Liu, Sung Keun Jung, Dong Joon Kim, Ann M Bode, Ki Won Lee, Zigang Dong
Intake of soy isoflavones is inversely associated with the risk of esophageal cancer. Numerous experimental results have supported the anti-cancer activity of soy isoflavones. The current study aimed to determine the anti-esophageal cancer activity of 6,7,4'-trihydroxyisoflavone (6,7,4'-THIF), a major metabolite of daidzein, which is readily metabolized in the human body. Notably, 6,7,4'-THIF inhibited proliferation and increased apoptosis of esophageal cancer cells. Based on a virtual screening analysis, Pin1 was identified as a target protein of 6,7,4'-THIF...
March 21, 2017: Cancer Prevention Research
https://www.readbyqxmd.com/read/28317851/mdhir-proteins-repress-anthocyanin-accumulation-by-interacting-with-the-mdjaz2-protein-to-inhibit-its-degradation-in-apples
#9
Ke-Qin Chen, Xian-Yan Zhao, Xiu-Hong An, Yi Tian, Dan-Dan Liu, Chun-Xiang You, Yu-Jin Hao
In higher plants, jasmonate ZIM-domain (JAZ) proteins negatively regulate the biosynthesis of anthocyanins by interacting with bHLH transcription factors. However, it is largely unknown if and how other regulators are involved in this process. In this study, the apple MdJAZ2 protein was characterized in regards to its function in the negative regulation of anthocyanin accumulation and peel coloration. MdJAZ2 was used as a bait to screen a cDNA library using the yeast two-hybrid method. The hypersensitive induced reaction (HIR) proteins, MdHIR2 and MdHIR4, were obtained from this yeast two-hybrid...
March 20, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28317380/systems-biology-reveals-ns4b-cyclophilin-a-interaction-a-new-target-to-inhibit-yfv-replication
#10
Alessandra Vidotto, Ana T S Morais, Milene R Ribeiro, Carolina C Pacca, Ana C B Terzian, Laura H V G Gil, Ronaldo Mohana-Borges, Philippe Gallay, Mauricio L Nogueira
Yellow fever virus (YFV) replication is highly dependent on host cell factors. YFV NS4B is reported to be involved in viral replication and immune evasion. Here interactions between NS4B and human proteins were determined using a GST pull-down assay and analyzed using 1-DE and LC-MS/MS. We present a total of 207 proteins confirmed using Scaffold 3 Software. Cyclophilin A (CypA), a protein that has been shown to be necessary for the positive regulation of flavivirus replication, was identified as a possible NS4B partner...
March 27, 2017: Journal of Proteome Research
https://www.readbyqxmd.com/read/28315004/alex1-a-novel-tumor-suppressor-gene-inhibits-gastric-cancer-metastasis-via-the-par-1-rho-gtpase-signaling-pathway
#11
Li Pang, Jian-Fang Li, Liping Su, Mingde Zang, Zhiyuan Fan, Beiqin Yu, Xiongyan Wu, Chen Li, Min Yan, Zheng-Gang Zhu, Bingya Liu
BACKGROUND: The ALEX is a novel member of the armadillo family and ALEX1 was reported to be reduced or even lost in multiple solid tumors. However, its expression profile and oncogenic role in gastric cancer (GC) remains largely unknown. METHODS: ALEX1 expression was detected in 161 GC samples by immunohistochemistry staining. NCI-N87 cells transfected by ALEX1 lentivirus vectors and MKN28 cells transfected by ALEX1 shRNA were used for biological function investigation...
March 17, 2017: Journal of Gastroenterology
https://www.readbyqxmd.com/read/28300839/bidirectional-transcription-of-linc00441-and-rb1-via-h3k27-modification-dependent-way-promotes-hepatocellular-carcinoma
#12
Junwei Tang, Yu Xie, Xiaoliang Xu, Yin Yin, Runqiu Jiang, Lei Deng, Zhongming Tan, Venkatanarayana Gangarapu, Jinhai Tang, Beicheng Sun
The retinoblastoma gene (RB1), a known tumor-suppressor gene (TSG), was decreased in multiple cancers including hepatocellular carcinoma (HCC). Here we focused on the bidirectional transcripted long noncoding RNA (Linc00441) with neighbor gene RB1 to investigate whether Linc00441 is involved in the suppression of RB1 in HCC. We found that aberrant upregulated intranuclear Linc00441 was reversely correlated with RB1 expression in human HCC samples. The gain- and loss-of-function investigation revealed that Linc00441 could promote the proliferation of HCC cells in vitro and in vivo with an apoptosis suppression and cell cycle rearrangement...
March 16, 2017: Cell Death & Disease
https://www.readbyqxmd.com/read/28299530/in-depth-characterization-of-the-homodimerization-domain-of-the-transcription-factor-thap1-and-dystonia-causing-mutations-therein
#13
Alev Richter, Ronja Hollstein, Eva Hebert, Franca Vulinovic, Juliane Eckhold, Alma Osmanovic, Reinhard Depping, Frank J Kaiser, Katja Lohmann
Mutations in the THAP1 gene encoding the transcription factor THAP1 have been shown to cause DYT6 dystonia. THAP1 contains a highly conserved THAP zinc finger at its N-terminal region which allows specific binding to its target sequences as well as a coiled-coil domain (amino acids 139-190) towards its C-terminus postulated as a protein-protein-binding motif. While several DYT6-causing mutations within the THAP domain were shown to decrease THAP1 activity in transcriptional regulation and DNA-binding, the role of mutations within the coiled-coil domain is rather unknown...
March 15, 2017: Journal of Molecular Neuroscience: MN
https://www.readbyqxmd.com/read/28298474/tumor-protein-d52-expression-is-post-transcriptionally-regulated-by-t-cell-intercellular-antigen-tia-1-and-tia-related-protein-via-mrna-stability
#14
Hiromi Motohashi, Yoshiki Mukudai, Chihiro Ito, Kosuke Kato, Toshikazu Shimane, Seiji Kondo, Tatsuo Shirota
Although tumor protein D52 (TPD) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than TPD53 and 54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation...
March 15, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28295592/knockdown-of-long-non-coding-rna-zfas1-protects-cardiomyocytes-against-acute-myocardial-infarction-via-anti-apoptosis-by-regulating-mir-150-crp
#15
Tao Wu, Dan Wu, Qinghua Wu, Bing Zou, Xiao Huang, Xiaoshu Cheng, Yanqing Wu, Kui Hong, Ping Li, Renqiang Yang, Yunde Li, Yingzhang Cheng
BACKGROUND: ZFAS1 is one of cardiac-specific or cardiac-related lncRNAs. This study was to explore the functional involvement of ZFAS1 and its regulatory role in AMI. METHODS: In this study, the models of AMI rat and myocardial cell cultured under hypoxia were made. The expression of ZFAS1 and miR-150 of myocardial infarction tissue or cardiac myocytes was determined by quantitative real time PCR. The regulatory role of ZFAS1 on miR-150 was examined by RNA pull down assay...
March 10, 2017: Journal of Cellular Biochemistry
https://www.readbyqxmd.com/read/28293843/analysis-of-ethylene-receptor-interactions-by-co-immunoprecipitation-assays
#16
Zhiyong Gao, G Eric Schaller
Ethylene receptors are predominantly localized to the endoplasmic reticulum (ER) membrane, and coordinate ethylene signal output through protein-protein interactions with each other and additional signaling components. Here, we describe a co-immunoprecipitation (Co-IP) assay based on the use of the Tandem Affinity Purification (TAP) tag to examine the interactions of ethylene receptors in plant extracts. Human IgG-agarose beads are used to pull down TAP-tagged versions of the protein of interest from detergent extracts of Arabidopsis membranes, and the precipitate then is analyzed immunologically for co-purification of the ethylene receptors...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28293543/the-tudor-staphylococcal-nuclease-protein-of-entamoeba-histolytica-participates-in-transcription-regulation-and-stress-response
#17
Javier Cázares-Apátiga, Christian Medina-Gómez, Bibiana Chávez-Munguía, Mercedes Calixto-Gálvez, Esther Orozco, Carlos Vázquez-Calzada, Aarón Martínez-Higuera, Mario A Rodríguez
Entamoeba histolytica is the protozoa parasite responsible of human amoebiasis, disease that causes from 40,000 to 100,000 deaths annually worldwide. However, few are known about the expression regulation of molecules involved in its pathogenicity. Transcription of some virulence-related genes is positively controlled by the cis-regulatory element named URE1. Previously we identified the transcription factor that binds to URE1, which displayed a nuclear and cytoplasmic localization. This protein belongs to the Tudor Staphyococcal nuclease (TSN) family, which in other systems participates in virtually all pathways of gene expression, suggesting that this amoebic transcription factor (EhTSN; former EhURE1BP) could also play multiple functions in E...
2017: Frontiers in Cellular and Infection Microbiology
https://www.readbyqxmd.com/read/28290451/development-of-sh2-probes-and-pull-down-assays-to-detect-pathogen-induced-site-specific-tyrosine-phosphorylation-of-the-tlr-adaptor-scimp
#18
Lin Luo, Samuel J Tong, Adam A Wall, Tatiana Khromykh, Matthew J Sweet, Jennifer L Stow
Protein tyrosine phosphorylation guides many molecular interactions for cellular functions. SCIMP is a transmembrane adaptor protein (TRAP) family member that mediates selective proinflammatory cytokine responses generated by pathogen-activated Toll-like receptor (TLR) pathways in macrophages. TLR activation triggers SCIMP phosphorylation and selective phosphorylation of distinct tyrosine residues on this adaptor offers the potential for regulating or biasing inflammatory responses. To analyze site-specific phosphorylation events, we developed three probes based on the SH2 domains of known SCIMP effectors, and used them for pull-downs from macrophage extracts...
March 14, 2017: Immunology and Cell Biology
https://www.readbyqxmd.com/read/28286910/the-molecular-mechanism-underlying-anthocyanin-metabolism-in-apple-using-the-mdmyb16-and-mdbhlh33-genes
#19
Haifeng Xu, Nan Wang, Jingxuan Liu, Changzhi Qu, Yicheng Wang, Shenghui Jiang, Ninglin Lu, Deyun Wang, Zongying Zhang, Xuesen Chen
MdMYB16 forms homodimers and directly inhibits anthocyanin synthesis via its C-terminal EAR repressor. It weakened the inhibitory effect of MdMYB16 on anthocyanin synthesis when overexpressing MdbHLH33 in callus overexpressing MdMYB16. MdMYB16 could interact with MdbHLH33. Anthocyanins are strong antioxidants that play a key role in the prevention of cardiovascular disease, cancer, and diabetes. The germplasm of Malus sieversii f. neidzwetzkyana is important for the study of anthocyanin metabolism. To date, only limited studies have examined the negative regulatory mechanisms underlying anthocyanin synthesis in apple...
March 12, 2017: Plant Molecular Biology
https://www.readbyqxmd.com/read/28277615/transcription-factor-ap-2%C3%AE-suppresses-cervical-cancer-cell-proliferation-by-promoting-the-degradation-of-its-interaction-partner-%C3%AE-catenin
#20
Fangmei Wang, Wenhuan Huang, Xiang Hu, Cheng Chen, Xinxin Li, Junlu Qiu, Zhongheng Liang, Jianmei Zhang, Limin Li, Xiaoqing Wang, Xiaofeng Ding, Shuanglin Xiang, Jian Zhang
Transcription factor AP-2β mediates the transcription of a number of genes implicated in mammalian development, cell proliferation and carcinogenesis. Although the expression pattern of AP-2β has been analyzed in cervical cancer cell lines, the functions and molecular mechanism of AP-2β are unknown. Here, we found that AP-2β significantly inhibits TCF/LEF reporter activity. Moreover, AP-2β and β-catenin interact both in vitro through GST pull-down assays and in vivo by co-immunoprecipitation. We further identified the interaction regions to the DNA-binding domain of AP-2β and the 1-9 Armadillo repeats of β-catenin...
March 9, 2017: Molecular Carcinogenesis
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