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Proximity ligation assay

Christine Bassila, Rose Ghemrawi, Justine Flayac, D Sean Froese, Matthias R Baumgartner, Jean-Louis Guéant, David Coelho
An increasing number of studies indicate that each step of the intracellular processing of vitamin B12 or cobalamin (Cbl) involves protein-protein interactions. We have previously described a novel interaction between methionine synthase (MS) and MMACHC and its effect on the regulation of MMACHC activity. Our goal is to further characterize the interactions of MS with other potential partners in a so-called MS interactome. We dissected the interactions and their alterations by co-immunoprecipitation and DuoLink proximity ligation assays in fibroblasts with cblG, cblE, and cblC genetic defects affecting respectively the expression of MS, methionine synthase reductase (MSR) and MMACHC and in HepG2 cells transfected with corresponding siRNAs...
October 19, 2016: Biochimica et Biophysica Acta
Jing Jiao, Rongxiang Han, Wayne W Hancock, Ulf H Beier
Determining protein acetylation by immunoprecipitation and immunoblotting can be challenging, especially if the tissue of interest is low in quantity, and when good quality acetylation site-specific antibodies are not available. Proximity ligation assays allow a sensitive and quantitative method to assess Foxp3 acetylation in regulatory T cells, with as little as 1.5 × 10(5) cells within two days turnaround time. This method is of potential use in other similar scenarios, when post-translational modifications of a protein of interest need to be determined with only a small amount of sample and in the absence of specific antibodies that can assess the post-translational modification in the protein of interest...
2017: Methods in Molecular Biology
Katharina Wolf, Susanne Strand
Generation of primary cell culture of hepatocytes by mouse liver perfusion (MLP) combines the advantages of in vivo and in vitro models. It provides hepatocytes that grow under physiological conditions in mice, with the genotype of the whole organism or a specific gene knockout. In contrast to immortalized cell cultures, primary murine hepatocytes (pmHep) are non-cancerous cells with a limited lifespan but still amenable to classical in vitro methods such as treatment with drugs, small molecule inhibitors, and agonistic/antagonistic antibodies of surface receptors as well as transfection...
2017: Methods in Molecular Biology
Jian Shi, Francesc Miralles, Lutz Birnbaumer, William A Large, Anthony P Albert
In vascular smooth muscle cells (VSMCs), stimulation of TRPC1-based SOCs mediate Ca(2+) entry pathways which regulate contractility, proliferation and migration. It is therefore important to understand how these channels are activated. Studies have shown that stimulation of TRPC1-based SOCs requires Gαq/PLCβ1 activities and PKC phosphorylation, but it is unclear how store depletion stimulates this gating pathway. The present work examines this issue by focusing on the role of STIM1, an endo/sarcoplasmic reticulum Ca(2+) sensor...
October 18, 2016: Journal of Physiology
Eun-Kyung Kwon, Chan-Ki Min, Yuri Kim, Jae-Won Lee, Abdimadiyeva Aigerim, Sebastian Schmidt, Hyun-Jun Nam, Seong Kyu Han, Kuglae Kim, Jeong Seok Cha, Hoyoung Kim, Sanguk Kim, Hyun-Soo Cho, Myung-Sik Choi, Nam-Hyuk Cho
Members of the herpesviral family use multiple strategies to hijack infected host cells and exploit cellular signaling for their pathogenesis and latent infection. Among the most intriguing weapons in the arsenal of pathogenic herpesviruses are the constitutively active virally-encoded G protein-coupled receptors (vGPCRs). Even though vGPCRs contribute to viral pathogenesis such as immune evasion and proliferative disorders, the molecular details of how vGPCRs continuously activate cellular signaling are largely unknown...
October 15, 2016: Biochimica et Biophysica Acta
Husni Elbahesh, Silke Bergmann, Charles J Russell
Influenza A viruses (IAVs) cause numerous pandemics and yearly epidemics resulting in ~500,000 annual deaths globally. IAV modulates cellular signaling pathways at every step of the infection cycle. Focal adhesion kinase (FAK) has been shown to play a critical role in endosomal trafficking of influenza A viruses, yet it is unclear how FAK kinase activity regulates IAV replication. Using mini-genomes derived from H1N1, H5N1 and H7N9 viruses, we dissected RNA replication by IAVs independent of viral entry or release...
October 13, 2016: Virology
Ting Yuan, Weitong Yao, Kenzo Tokunaga, Rongge Yang, Binlian Sun
BACKGROUND: Several members of the TRIM family have been implicated in antiviral defense. Our previous report showed that human TRIM11 potently inhibited HIV-1 transduction by reducing the viral reverse transcripts. These results prompted us to examine the effect of TRIM11 on HIV-1 uncoating, which is closely related to viral reverse transcription. RESULTS: Using a combination of in vitro binding and in situ proximity ligation assay, we showed that TRIM11 could interact with HIV-1 capsid...
October 13, 2016: Retrovirology
M-U-D Lone, K S Baghel, R K Kanchan, R Shrivastava, S A Malik, B N Tewari, C Tripathi, M P S Negi, V K Garg, M Sharma, M L B Bhatt, S Bhadauria
Augmented reactive oxygen species levels consequential to functional alteration of key mitochondrial attributes contribute to carcinogenesis, either directly via oxidative DNA damage infliction or indirectly via activation of oncogenic signaling cascades. We previously reported activation of a key oncogenic signaling cascade via mammalian target of rapamycin (mTOR) signaling complex-2 (mTORC2) owing to estrogen receptor (ER-α)-dependent augmentation of O2(.-) within the mitochondria of 17-β-estradiol (E2)-stimulated breast cancer cells...
October 10, 2016: Oncogene
Vera Dugina, Irina Alieva, Natalya Khromova, Igor Kireev, Peter W Gunning, Pavel Kopnin
Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of β- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not β-cytoplasmic actin via the microtubule +TIPs protein EB1...
September 24, 2016: Oncotarget
Liza Löf, Tonge Ebai, Louise Dubois, Lotta Wik, K Göran Ronquist, Olivia Nolander, Emma Lundin, Ola Söderberg, Ulf Landegren, Masood Kamali-Moghaddam
Flow cytometry is a powerful method for quantitative and qualitative analysis of individual cells. However, flow cytometric analysis of extracellular vesicles (EVs), and the proteins present on their surfaces has been hampered by the small size of the EVs - in particular for the smallest EVs, which can be as little as 40 nm in diameter, the limited number of antigens present, and their low refractive index. We addressed these limitations for detection and characterization of EV by flow cytometry through the use of multiplex and multicolor in situ proximity ligation assays (in situ PLA), allowing each detected EV to be easily recorded over background noise using a conventional flow cytometer...
September 29, 2016: Scientific Reports
A D Rao, Y Liu, C C Hsu, A Parekh, L M Rosati, K Ng, A Hacker-Prietz, L Zheng, T M Pawlik, D A Laheru, E M Jaffee, M J Weiss, D T Le, R H Hruban, A De Jesus-Acosta, C L Wolfgang, D T Chang, A C Koong, J M Herman
No abstract text is available yet for this article.
October 1, 2016: International Journal of Radiation Oncology, Biology, Physics
Ill-Min Chung, Sarada Ketharnathan, Seung-Hyun Kim, Muthu Thiruvengadam, Mari Kavitha Rani, Govindasamy Rajakumar
Proximity ligation assays such as circularized chromosome conformation capture and high-throughput chromosome capture assays have shed light on the structural organization of the interphase genome. Functional topologically associating domains (TADs) that constitute the building blocks of genomic organization are disrupted and reconstructed during the cell cycle. Epigenetic memory, as well as the sequence of chromosomes, regulate TAD reconstitution. Sub-TAD domains that are invariant across cell types have been identified, and contacts between these domains, rather than looping, are speculated to drive chromatin folding...
2016: Genes
Kalu U E Ogbureke, Komal Koli, Geetu Saxena
We recently reported the expression of matrix metalloproteinase 20 (MMP20), hitherto thought to be tooth specific, in the metabolically active ductal epithelial cells of human salivary glands. Furthermore, our report indicated that MMP20 co-expressed and potentially interacts with dentin sialophosphoprotein (DSPP), a member of the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Our earlier reports have shown the co-expression of three MMPs, MMP2, MMP3, and MMP9, with specific members of the SIBLING family: bone sialoprotein, osteopontin, and dentin matrix protein 1, respectively...
October 2016: Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society
Michaela Frolikova, Natasa Sebkova, Lukas Ded, Katerina Dvorakova-Hortova
The acrosome reaction (AR) is a process of membrane fusion and lytic enzyme release, which enables sperm to penetrate the egg surroundings. It is widely recognized that specific sperm proteins form an active network prior to fertilization, and their dynamic relocation is crucial for the sperm-egg fusion. The unique presence of the membrane cofactor protein CD46 in the sperm acrosomal membrane was shown, however, its behaviour and connection with other sperm proteins has not been explored further. Using super resolution microscopy, we demonstrated a dynamic CD46 reorganisation over the sperm head during the AR, and its interaction with transmembrane protein integrins, which was confirmed by proximity ligation assay...
September 26, 2016: Scientific Reports
C Zurla, J Jung, E L Blanchard, P J Santangelo
RNA binding proteins (RBP) and small RNAs regulate the editing, localization, stabilization, translation, and degradation of ribonucleic acids (RNAs) through their interactions with specific cis-acting elements within target RNAs. Here, we describe a novel method to detect protein-mRNA interactions, which combines FLAG-peptide modified, multiply-labeled tetravalent RNA imaging probes (FMTRIPs) with proximity ligation (PLA), and rolling circle amplification (RCA). This assay detects native RNA in a sequence specific and single RNA sensitive manner, and PLA allows for the quantification and localization of protein-mRNA interactions with single-interaction sensitivity...
2017: Methods in Molecular Biology
Wei Zhang, Mingyi Xie, Mei-Di Shu, Joan A Steitz, Daniel DiMaio
The proximity ligation assay (PLA) is an immune staining method that detects protein-protein interactions in fixed cells. We describe here RNA-PLA, a simple adaptation of this technology that allows the detection of specific RNA-protein interactions in fixed cells by using a DNA oligonucleotide that hybridizes to a target RNA in combination with an antibody that recognizes the protein bound to the target RNA. Stable and transient RNA-protein interactions can be readily detected by generation of a fluorescent signal in discrete compartments in intact fixed cells with high specificity...
September 22, 2016: RNA
Manisha Sharma, Cara Jamieson, Christina Lui, Beric R Henderson
β-catenin is a key mediator of Wnt signaling and its deregulated nuclear accumulation can drive cancer progression. While the central armadillo (Arm) repeats of β-catenin stimulate nuclear entry, the N- and C-terminal "tail" sequences are thought to regulate turnover and transactivation. We show here that the N- and C-tails are also potent transport sequences. The unstructured tails of β-catenin, when individually fused to a GFP-reporter, could enter and exit the nucleus rapidly in live cells. Proximity ligation assays and pull-down assays identified a weak interaction between the tail sequences and the FG-repeats of nucleoporins, consistent with a possible direct translocation of β-catenin through the nuclear pore complex...
September 19, 2016: Experimental Cell Research
Syeda Ridita Sharif, Ariful Islam, Il Soo Moon
N-acetyl-D-glucosamine kinase (GlcNAc kinase or NAGK) primarily catalyzes phosphoryl transfer to GlcNAc during amino sugar metabolism. Recently, it was shown NAGK interacts with dynein light chain roadblock type 1 (DYNLRB1) and upregulates axo-dendritic growth, which is an enzyme activity-independent, non-canonical structural role. The authors examined the distributions of NAGK and NAGK-dynein complexes during the cell cycle in HEK293T cells. NAGK was expressed throughout different stages of cell division and immunocytochemistry (ICC) showed NAGK was localized at nuclear envelope, spindle microtubules (MTs), and kinetochores (KTs)...
September 2016: Molecules and Cells
Dorota Maszczak-Seneczko, Paulina Sosicka, Teresa Olczak, Mariusz Olczak
In situ proximity ligation assay (PLA) is a novel, revolutionary technique that can be employed to visualize protein complexes in fixed cells and tissues. This approach enables demonstration of close (i.e., up to 40 nm) proximity between any two proteins of interest that can be detected using a pair of specific antibodies that have been raised in distinct species. Primary antibodies bound to the target proteins are subsequently recognized by two PLA probes, i.e., secondary antibodies conjugated with oligonucleotides that anneal when brought into close proximity in the presence of two connector oligonucleotides and a DNA ligase forming a circular DNA molecule...
2016: Methods in Molecular Biology
Umut Sahin, Florence Jollivet, Caroline Berthier, Hugues de Thé, Valérie Lallemand-Breitenbach
Sumoylation is a posttranslational process essential for life and concerns a growing number of crucial proteins. Understanding the influence of this phenomenon on individual proteins or on cellular pathways in which they function has become an intense area of research. A critical step in studying protein sumoylation is to detect sumoylated forms of a particular protein. This has proven to be a challenging task for a number of reasons, especially in the case of endogenous proteins and in vivo studies or when studying rare cells such as stem cells...
2016: Methods in Molecular Biology
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