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Pull down assay

Xiao-Ming Wang, Yang Liu, Yu-Xia Fan, Zheng Liu, Qing-Ling Yuan, Meng Jia, Geng Zu-Shi, Ling Gu, Xiu-Bo Lu
Background LncRNA PTCSC3 is a tumor suppressor in thyroid cancer, and its role in drug resistance of anaplastic thyroid cancer (ATC) to chemotherapy drug doxorubicin was investigated in this study. Methods Expression of RNA and protein was analyzed by qRT-PCR and western blot, respectively. Flow cytometry was used to analyze the expression rate of CD133+ cells. The endogenous expression of related genes was modulated by recombinant plasmids and cell transfection. Combination condition and interaction between PTCSC3 and STAT3 were determined by RIP and RNA pull-down assay, respectively...
March 21, 2018: Cancer Biology & Therapy
Qin Chen, Rong Deng, Xian Zhao, Haihua Yuan, Hailong Zhang, Jinzhuo Dou, Ran Chen, Hui Jin, Yanli Wang, Jian Huang, Jianxiu Yu
BACKGROUND/AIMS: An increasing number of studies have linked <unterline>e</unterline>rythropoietin-<unterline>p</unterline>roducing <unterline>h</unterline>epatocellular carcinoma (Eph) family receptor tyrosine kinases to cancer progression. However, little knowledge is available about the regulation of their functions in cancer. METHODS: SUMOylation was analyzed by performing Ni2+-NTA pull-down assay and immunoprecipitation. Cell proliferation, anchorage-independent growth, and tumorigenesis in vivo were examined by cell counting kit-8, soft agar colony formation assay, and a xenograft tumor mouse model, respectively...
March 13, 2018: Cellular Physiology and Biochemistry
Kaixuan Zeng, Xiaoxiang Chen, Mu Xu, Xiangxiang Liu, Xiuxiu Hu, Tao Xu, Huiling Sun, Yuqin Pan, Bangshun He, Shukui Wang
Mounting evidences indicate that circular RNAs (circRNAs) have a vital role in human diseases, especially cancers. More recently, circHIPK3, a particularly abundant circRNA, was proposed to be involved in tumorigenesis. However, its role in colorectal cancer (CRC) has not been explored. In this study, we found circHIPK3 was significantly upregulated in CRC tissues and cell lines, at least in part, due to c-Myb overexpression and positively correlated with metastasis and advanced clinical stage. Moreover, Cox multivariate survival analysis showed that high-level expression of circHIPK3 was an independent prognostic factor of poor overall survival (OS) in CRC (hazard ratio [HR] = 2...
March 16, 2018: Cell Death & Disease
Memi Muto, Wataru Kamitani, Mizuki Sakai, Minato Hirano, Shintaro Kobayashi, Hiroaki Kariwa, Kentaro Yoshii
Tick-borne encephalitis virus (TBEV) causes severe neurological disease, but the pathogenetic mechanism is unclear. The conformational structure of the 3'-untranslated region (UTR) of TBEV is associated with its virulence. We tried to identify host proteins interacting with the 3'-UTR of TBEV. Cellular proteins of HEK293T cells were co-precipitated with biotinylated RNAs of the 3'-UTR of low- and high-virulence TBEV strains and subjected to mass spectrometry analysis. Fifteen host proteins were found to bind to the 3'-UTR of TBEV, four of which- cold shock domain containing-E1 (CSDE1), spermatid perinuclear RNA binding protein (STRBP), fragile X mental retardation protein (FMRP), and interleukin enhancer binding factor 3 (ILF3)-bound specifically to that of the low-virulence strain...
March 12, 2018: Virus Research
Amit Gaba, Lisanework E Ayalew, Amrutlal Patel, Pankaj Kumar, Suresh K Tikoo
Adenovirus protein VIII appears to connect core with the inner surface of the adenovirus capsid. Since protein- protein interactions are central to virus replication, identification of proteins interacting with protein VIII may help in understanding their role in adenovirus infection. Our yeast two hybrid assay indicated that protein VIII interacts with eukaryotic initiation factor 6 (eIF6). These findings were confirmed by GST-pull down assay, bimolecular fluorescent complementation assay and co-immunoprecipitation assay in plasmid DNA transfected and BAdV-3 infected cells...
March 15, 2018: Cellular Microbiology
Xuren Gao, Jian Ge, Weiyi Li, Wangchen Zhou, Lei Xu
Background: Resveratrol (RSV) has been reported to stimulate osteoblast differentiation in which Wnt/β-catenin signaling pathway played a crucial role. However, whether and how RSV activated Wnt/β-catenin pathway in osteogenic differentiation still remained elusive. Methods: In vivo polymethylmethacrylate (PMMA) particle-induced osteolysis (PIO) mouse model and in vitro PMMA particle-stimulated mouse mesenchymal stem cells (mMSCs) experiments were established...
2018: Cell & Bioscience
Min Sha, Mei Lin, Jia Wang, Jun Ye, Jie Xu, Ning Xu, Junxing Huang
BACKGROUND: The objective of this study was to investigate the role and mechanism of long non-coding RNA MIAT in gastric cancer (GC). METHODS: Real-time PCR was used to determine MIAT level in 120 GC tissues, and in two gastric cancer cell lines. The clinicopathological characteristics of MIAT in GC patients were analyzed. Small interfering RNA specific for MIAT (si-MIAT) and lentivector for si-MIAT was performed to down-regulate MIAT expression in GC cells and in animal tumor model, respectively...
March 14, 2018: Journal of Experimental & Clinical Cancer Research: CR
Mattia Laffranchi, Romina Berardelli, Riccardo Ronzoni, David A Lomas, Annamaria Fra
The most common genotype associated with severe α1-antitrypsin deficiency (AATD) is the Z homozygote. The Z variant (Glu342Lys) of α1-antitrypsin (AAT) undergoes a conformational change and is retained within the endoplasmic reticulum (ER) of hepatocytes leading to the formation of ordered polymeric chains and inclusion bodies. Accumulation of mutated protein predisposes to cirrhosis whilst plasma AAT deficiency leads to emphysema. Increased risk of liver and lung disease has also been reported in heterozygous subjects who carry Z in association with the milder S allele (Glu264Val) or even with wild-type M...
March 10, 2018: Human Molecular Genetics
Chieun Song, Jimin Lee, Taeyoon Kim, Jong Chan Hong, Chae Oh Lim
Under normal growth conditions, Arabidopsis VOZ1 interacts with DREB2C and acts as a transcriptional repressor by reducing DNA binding of DREB2C. Under heat stress conditions, VOZ1 is degraded by ubiquitination, and DREB2C, which is freed from VOZ1, functions as a transcription activator. To investigate the mechanism by which the DEHYDRATION-RESPONSIVE ELEMENT-BINDING FACTOR 2C (DREB2C)-dependent signaling cascade regulates heat stress (HS) responses, we performed a yeast two-hybrid screening using the DREB2C APETALA2 (AP2) DNA-binding domain as the bait against a cDNA library derived from Arabidopsis...
March 13, 2018: Planta
Zhiqiang Duan, Haixu Xu, Xinqin Ji, Jiafu Zhao, Houqiang Xu, Yan Hu, Shanshan Deng, Shunlin Hu, Xiufan Liu
The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays...
March 13, 2018: Virulence
Shifeng Ling, Mingyang Luo, Shengnan Jiang, Jiayu Liu, Chunying Ding, Qinghuan Zhang, Huancheng Guo, Wenjie Gong, Changchun Tu, Jinfu Sun
Classical swine fever virus (CSFV) nonstructural protein NS5A is a multifunctional protein functioning in regulation of viral genome replication, protein translation and assembly by interaction with viral or host proteins. Here, heat shock protein 27 (Hsp27) has been identified as a novel binding partner of NS5A by using His tag "pull down" coupled with shotgun LC-MS/MS, with interaction of both proteins further confirmed by co-immunoprecipitation and laser confocal assays. In PK-15 cells, silencing of Hsp27 expression by siRNA enhanced CSFV replication, and upregulation of Hsp27 inhibited viral proliferation...
March 8, 2018: Virology
Chang-Biao Chi, YaJun Tang, Jiahai Zhang, Ya-Nan Dai, Mohnad Abdalla, Yuxing Chen, Cong-Zhao Zhou
The yeast Sacchromyces cerevisiae monothiol glutaredoxin Grx3 plays a key role in cellular defense against oxidative stress and more importantly, cooperates with BolA-like iron repressor of activation protein Fra2 to regulate the localization of the iron-sensing transcription factor Aft2. The interplay among Grx3, Fra2 and Aft2 responsible for the regulation of iron homeostasis has not been clearly described. Here we solved the crystal structures of the Trx domain (Grx3Trx ) and Grx domain (Grx3Grx ) of Grx3 in addition to the solution structure of Fra2...
March 7, 2018: Journal of Molecular Biology
Lei Gu, Lie-Sheng Lu, Dong-Lei Zhou, Zhong-Chen Liu
Long noncoding RNAs (lncRNAs) have emerged as regulators in a variety of biological processes, including carcinogenesis in human cancer. UCA1 has been reported to be upregulated in gastric cancer (GC); however, the underlying functional roles of UCA1 in GC have not been established. In the current study, we showed that UCA1 is significantly higher in GC tissues and cells compared with adjacent normal tissues and a gastric epithelium cell line, respectively. Higher UCA1 expression was associated with lymph node metastasis, TNM stage, and poor overall survival (OS) in GC patients...
March 8, 2018: Cancer Medicine
Yan Liang, Xuedan Chen, Yuanyuan Wu, Juan Li, Shixin Zhang, Kai Wang, Xingying Guan, Kang Yang, Yun Bai
Esophageal squamous cell carcinoma (ESCC) is the main subtype of esophageal cancer. Long noncoding RNAs (lncRNAs) are thought to play a critical role in cancer development. Recently, lncRNA CASC9 was shown to be dysregulated in many cancer types, but the mechanisms whereby this occurs remain largely unknown. In this study, we found that CASC9 was significantly upregulated in ESCC tissues, with further analysis revealing that elevated CASC9 expression was associated with ESCC prognosis and metastasis. Furthermore, we found that CASC9 knockdown significantly repressed ESCC migration and invasion in vitro and metastasis in nude mice in vivo...
March 6, 2018: Cell Death and Differentiation
Ji Pang, Pengcheng Jiang, Ying Wang, Lu Jiang, Hai Qian, Yan Tao, Ruxia Shi, Jizong Gao, Yongchang Chen, Yan Wu
BACKGROUND: The recurrence, metastasis and poor prognosis are important characteristics of ovarian carcinoma (OC), which are associated with exfoliation of cells from the primary tumor and colonization of the cells in pelvic cavity. On the other hand, the life quality of the patients undergoing surgical resection of OC was influenced by postoperative adhesions. Therefore, preventing postoperative implant tumor and adhesion may be effective methods to improve OC treatment. HyaRegen Gel, a cross-linked hyaluronan gel (CHAG), has been widely used as an anti-adhesive agent following pelvic operation in clinic...
March 6, 2018: Journal of Ovarian Research
Xiao-Lu Xie, Xue-Jiao Chang, Yan Gao, Dong-Li Li, Ling-Ke Liu, Man-Jun Liu, Ke-Jian Wang, Hai-Peng Liu
Influenza A virus non-structural-1A binding protein (named as Ns1abp) was originally identified as a host protein from human that bound to the viral NS-1 protein. In our previous study, the expression of an Ns1abp-like gene (denoted as CqNs1abp-like gene) was found to be up-regulated in a transcriptome library from the haematopoietic tissue (Hpt) cells of red claw crayfish Cherax quadricarinatus post white spot syndrome virus (WSSV) infection. To elucidate the role of CqNs1abp-like gene involved in WSSV infection, we cloned the CqNs1abp-like gene in which the open reading frame was 2232 bp, encoding 743 amino acids with two typical domains of one BTB (Broad-Complex, Tramtrack and Bric a brac) domain at N-terminal and six Kelch domains at C-terminal...
March 3, 2018: Developmental and Comparative Immunology
Jie Ren, Tingting Lian, Lili Shao, Yuanjun Liu, Quanzhong Liu
Chlamydia trachomatis (C. trachomatis) is the most common cause of bacterial sexually transmitted infections. The effect of antibiotic treatment is not satisfactory, and there is currently no vaccine to prevent C. trachomatis infection. Our results showed that Chlamydia virus CPG1 capsid protein Vp1 treatment significantly inhibited C. trachomatis growth in cell culture, and the inclusion numbers of different C. trachomatis serotypes were decreased. In addition, we conducted a preliminary investigation of the possible mechanisms behind the Vp1 inhibition effects and C...
March 6, 2018: Canadian Journal of Microbiology
Sushil Kumar, Pratibha Choudhary, Mansi Gupta, Utpal Nath
In plants, endogenous and environmental signals such as light control the timing of the transition to flowering. Two phytochrome B-interacting transcription factors, VASCULAR PLANT ONE-ZINC FINGER1 (VOZ1) and VOZ2 redundantly promote flowering in Arabidopsis thaliana. In the voz1 voz2 mutant the expression of FLOWERING LOCUS C (FLC) was up-regulated and that of FLOWERING LOCUS T (FT) was down-regulated, which was proposed to be the cause of late flowering in voz1 voz2. However, the detailed mechanism by which the VOZ genes promote flowering is not well understood...
March 5, 2018: Plant Physiology
Silvia Belluti, Valentina Semeghini, Valentina Basile, Giovanna Rigillo, Valentina Salsi, Filippo Genovese, Diletta Dolfini, Carol Imbriano
The heterotrimeric NF-Y complex is a pioneer factor that binds to CCAAT-genes and regulates their transcription. NF-Y cooperates with multiple transcription factors and co-regulators in order to positively or negatively influence gene transcription. The recruitment of NF-Y to CCAAT box is significantly enriched in cancer-associated gene promoters loci and positively correlates with malignancy. NF-Y subunits, in particular the DNA-binding subunit NF-YA and the histone-fold subunit NF-YC, appear overexpressed in specific types of cancer...
March 2, 2018: Biochimica et Biophysica Acta
Kristyna Burglova, Gabriela Rylova, Athanasios Markos, Hana Prichystalova, Miroslav Soural, Marek Petracek, Martina Medvedikova, Gracian Tejral, Bruno Sopko, Pavel Hradil, Petr Dzubak, Marian Hajduch, Jan Hlavac
With use of computational modelling we have identified interaction site of the contraceptive drug Gamendazole, described previously as a ligand for Eukaryotic Translation Elongation Factor (eEF1A1) and found it as the potential target site for derivatives of 2-phenyl-3-hydroxy-4(1H)-quinolinones (3-HQs) with anticancer activity. The interaction of this class of compounds with this protein inside the cancer cells was then confirmed via pull-down assay. With this knowledge, we designed and synthesized a new family of 3-HQs and confirmed, by isothermal titration calorimetry, that these compounds strongly bind to this protein...
March 2, 2018: Journal of Medicinal Chemistry
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