| 1. |
- Abramsson, Alexandra, 1973, et al.
(författare)
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Defective N-sulfation of heparan sulfate proteoglycans limits PDGF-BB binding and pericyte recruitment in vascular development
- 2007
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Ingår i: GENES & DEVELOPMENT. - : Cold Spring Harbor Laboratory. - 0890-9369 .- 1549-5477. ; 21:3, s. 316-331
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Tidskriftsartikel (refereegranskat)abstract
- During vascular development, endothelial platelet-derived growth factor B (PDGF-B) is critical for pericyte recruitment. Deletion of the conserved C-terminal heparin-binding motif impairs PDGF-BB retention and pericyte recruitment in vivo, suggesting a potential role for heparan sulfate (HS) in PDGF-BB function during vascular development. We studied the participation of HS chains in pericyte recruitment using two mouse models with altered HS biosynthesis. Reduction of N-sulfation due to deficiency in N-deacetylase/N-sulfotransferase-1 attenuated PDGF-BB binding in vitro, and led to pericyte detachment and delayed pericyte migration in vivo. Reduced N-sulfation also impaired PDGF-BB signaling and directed cell migration, but not proliferation. In contrast, HS from glucuronyl C5-epimerase mutants, which is extensively N- and 6-O-sulfated, but lacks 2-O-sulfated L-iduronic acid residues, retained PDGF-BB in vitro, and pericyte recruitment in vivo was only transiently delayed. These observations were supported by in vitro characterization of the structural features in HS important for PDGF-BB binding. We conclude that pericyte recruitment requires HS with sufficiently extended and appropriately spaced N-sulfated domains to retain PDGF-BB and activate PDGF receptor β (PDGFRβ) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.
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| 2. |
- Darmanis, Spyros, et al.
(författare)
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Self-assembly of proximity probes for flexible and modular proximity ligation assays
- 2007
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Ingår i: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 43:4, s. 443-450
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Tidskriftsartikel (refereegranskat)abstract
- Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.
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| 3. |
- Ericsson, Olle, et al.
(författare)
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A dual-tag microarray platform for high-performance nucleic acid and protein analyses
- 2008
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 36:8, s. e45-
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Tidskriftsartikel (refereegranskat)abstract
- DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 10(5). Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively.
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| 4. |
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| 5. |
- Gullberg, Mats, et al.
(författare)
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Cytokine detection by antibody-based proximity ligation
- 2004
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 101:22, s. 8420-4
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Efficient and precise detection techniques, along with extensive repertoires of specific binding reagents, will be needed to meet the challenges of proteome analyses. The recently established proximity ligation mechanism enables sensitive high-capacity protein measurements by converting the detection of specific proteins to the analysis of DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins and to form amplifiable tag sequences by ligation when brought in proximity. In our previous report, both the ligatable arms and the protein binders were DNA molecules. We now generalize the method by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences. Sufficient reagent for >100,000 proximity ligation assays can be prepared from 1 microg of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-microl samples, and we exemplify its utility in situations when only minute sample amounts are available.
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| 6. |
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| 7. |
- Gustafsdottir, Sigrun M., et al.
(författare)
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Detection of individual microbial pathogens by proximity ligation
- 2006
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 52:6, s. 1152-1160
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Nucleic acid amplification allows the detection of single infectious agents. Protein-based assays, although they provide information on ongoing infections, have substantially less detection sensitivity.METHODS: We used proximity ligation reactions to detect proteins on bacteria and virus particles via nucleic acid amplification. Antibodies recognizing viral or bacterial surface proteins were equipped with DNA strands that could be joined by ligation when several antibodies were bound in proximity to surface proteins of individual infectious agents.RESULTS: Detection sensitivities similar to those of nucleic acid-based detection reactions were achieved directly in infected samples for a parvovirus and an intracellular bacterium.CONCLUSIONS: This method enables detection of ligated DNA strands with good sensitivity by real-time PCR and could be of value for early diagnosis of infectious disease and in biodefense.
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| 8. |
- Gustafsdottir, Sigrun M., et al.
(författare)
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In vitro analysis of DNA-protein interactions by proximity ligation
- 2007
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 104:9, s. 3067-3072
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Tidskriftsartikel (refereegranskat)abstract
- Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro method for specific and sensitive solution-phase analysis of interactions between proteins and nucleic acids in nuclear extracts, based on the proximity ligation assay. The reagent consumption is very low, and the excellent sensitivity of the assay enables analysis of as few as 1-10 cells. We show that our results are highly reproducible, quantitative, and in good agreement with both EMSA and predictions obtained by using a motif finding software. This assay can be a valuable tool to characterize in-depth the sequence specificity of DNA-binding proteins and to evaluate effects of polymorphisms in known transcription factor binding sites.
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| 9. |
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| 10. |
- Gustafsdottir, Sigrun M, et al.
(författare)
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Use of Proximity Ligation to Screen for Inhibitors of Interactions between Vascular Endothelial Growth Factor A and Its Receptors
- 2008
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 54:7, s. 1218-1225
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Improved methods are required to screen drug candidates for their influences on protein interactions. There is also a compelling need for miniaturization of screening assays, with attendant reductions in reagent consumption and assay costs. METHODS: We used sensitive, miniaturized proximity ligation assays (PLAs) to monitor binding of vascular endothelial growth factor A (VEGF-A) to 2 of its receptors, VEGFR-1 and VEGFR-2. We measured the effects of proteins and low molecular weight compounds capable of disrupting these interactions and compared the results with those obtained by immunoblot analysis. We analyzed 6 different inhibitors: a DNA aptamer, a mixed DNA/RNA aptamer, a monoclonal VEGF-A neutralizing antibody, a monoclonal antibody directed against VEGFR-2, a recombinant competitive protein, and a low molecular weight synthetic molecule. RESULTS: The PLAs were successful for monitoring the formation and inhibition of VEGF-A-receptor complexes, and the results correlated well with those obtained by measuring receptor phosphorylation. The total PLA time is just 3 hours, with minimal manual work and reagent additions. The method allows evaluation of the apparent affinity [half-maximal inhibitory concentration (IC(50))] from a dose-response curve. CONCLUSIONS: The PLA may offer significant advantages over conventional methods for screening the interactions of ligands with their receptors. The assay may prove useful for parallel analyses of large numbers of samples in the screening of inhibitor libraries for promising agents. The technique provides dose-response curves, allowing IC(50) values to be calculated.
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