| 1. |
- Barbe, Laurent, et al.
(author)
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Toward a confocal subcellular atlas of the human proteome
- 2008
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In: Molecular and cellular proteomics. - 1535-9476 .- 1535-9484. ; 7:3, s. 499-508
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Journal article (peer-reviewed)abstract
- Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.
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| 2. |
- Berglund, Lisa, et al.
(author)
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A genecentric Human Protein Atlas for expression profiles based on antibodies
- 2008
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In: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 7:10, s. 2019-2027
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Research review (peer-reviewed)abstract
- An attractive path forward in proteomics is to experimentally annotate the human protein complement of the genome in a genecentric manner. Using antibodies, it might be possible to design protein-specific probes for a representative protein from every protein-coding gene and to subsequently use the antibodies for systematical analysis of cellular distribution and subcellular localization of proteins in normal and disease tissues. A new version (4.0) of the Human Protein Atlas has been developed in a genecentric manner with the inclusion of all human genes and splice variants predicted from genome efforts together with a visualization of each protein with characteristics such as predicted membrane regions, signal peptide, and protein domains and new plots showing the uniqueness (sequence similarity) of every fraction of each protein toward all other human proteins. The new version is based on tissue profiles generated from 6120 antibodies with more than five million immunohistochemistry-based images covering 5067 human genes, corresponding to approximately 25% of the human genome. Version 4.0 includes a putative list of members in various protein classes, both functional classes, such as kinases, transcription factors, G-protein-coupled receptors, etc., and project-related classes, such as candidate genes for cancer or cardiovascular diseases. The exact antigen sequence for the internally generated antibodies has also been released together with a visualization of the application-specific validation performed for each antibody, including a protein array assay, Western blot analysis, immunohistochemistry, and, for a large fraction, immunofluorescence-based confocal microscopy. New search functionalities have been added to allow complex queries regarding protein expression profiles, protein classes, and chromosome location. The new version of the protein atlas thus is a resource for many areas of biomedical research, including protein science and biomarker discovery.
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| 3. |
- Lerner, Ulf H, et al.
(author)
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Kinins and neuro-osteogenic factors
- 2008
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In: Principles of bone biology. 3rd edition. - San Diego : Acadamic Press. - 9780123738844 ; , s. 1025-57
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Book chapter (other academic/artistic)
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| 4. |
- Lerner, Ulf H, 1946-, et al.
(author)
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Skelettet vid hälsa och sjukdom
- 2008
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In: Tandläkartidningen. - 0039-6982. ; 100:5, s. 84-90
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Journal article (other academic/artistic)abstract
- Hur ombyggnaden av benvävnaden i käkar och skelett fungerar och hur processen påverkas vid sjukdomstillstånd som parodontit, benskörhet, tumörer med mera är föremål för omfattande forskning i Umeå.
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| 5. |
- Lundberg, Emma, et al.
(author)
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A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase
- 2007
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In: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 322:1-2, s. 40-49
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Journal article (peer-reviewed)abstract
- Fluorescently labeled antibodies are very important tools in cell biology, providing for specific and quantitative detection of antigens. To date, fluorophore labeling of antibodies has been performed in solution and has been limited by low-throughput methods requiring a substantial amount of pure antibody sample at a high concentration. We have developed a novel solid-phase labeling protocol for small amounts (i.e. micrograms) of antibodies with fluorescent dyes. Protein A affinity medium was used as solid support in a micropipette tip format. This solid-phase approach, including the advantage of the strong and specific interaction between Protein A and antibodies, allows for simultaneous purification, labeling and concentration of the antibody sample, making it possible to start with unpure antibody samples at low concentrations. We have optimized the protocol with regard to reaction pH, time, temperature and amount of amine reactive dye. In addition, we have evaluated the stability and activity of the labeled antibodies. To evaluate the reproducibility and robustness of this method we labeled eight antibodies with amine reactive fluorescent dyes followed by evaluation of antibody specificity on protein arrays. Interestingly, this gave an extremely high conformity in the degree of labeling, showing the robustness of the method. The solid-phase method also gave predictable and reproducible results and by varying the amount of reactive dye, the desired degree of labeling can easily be achieved. Antibodies labeled using this solid-phase method were similar in stability and activity to antibodies labeled in solution. This novel solid-phase antibody labeling method may also be applicable for other conjugation chemistries and labels, and has potential for throughput applications.
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| 6. |
- Lundberg, Emma, 1980-
(author)
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Bioimaging for analysis of protein expression in cells and tissues using affinity reagents
- 2008
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Doctoral thesis (other academic/artistic)abstract
- The detection and analysis of biomolecules, such as proteins, are of great interest since these molecules are fundamental for life and our health. Due to the complexity of biological processes, there is a great advantage of studying proteins in their natural context, for example by using bioimaging. The objective of this doctoral thesis has been to develop, implement and evaluate techniques for the use of proteinspecific affinity reagents in diverse bioimaging platforms for analysis of protein expression in situ in cells and tissues. To be able to visualize a desired protein in situ using affinity reagents, reporter labels are needed. A novel technique for labeling of antibodies on solid phase was developed. This method offers simultaneous purification, concentration and labeling of an antibody sample, giving highly predictable and reproducible results, in a miniaturized format. Another study demonstrates the use of an alternative affinity reagent, the Affibody molecule, in bioimaging as well as other immunoassays. As a relevant proof-of-principle, an Affibody molecule binding the HER2 receptor was site-specificly labeled and employed for analysis of HER2 protein expression in cells and tissue using immunofluorescence (IF), immunohistochemistry (IHC), immunoprecipitation and flow cytometry. Furthermore, it is shown how antibody-based bioimaging approaches can be applied for systematic analysis of protein expression in terms of subcellular localization and expression levels in cell lines. The systematic subcellular localization of nearly 500 proteins was performed using IF and confocal microscopy. Global analysis of expression levels of nearly 2000 proteins in a panel of cell lines using IHC and automated image analysis, revealed that most proteins are expressed in a cell size dependent manner. Two normalization approaches were evaluated and found to allow for protein profiling across the panel of morphologically diverse cells, revealing patterns of protein over- and underexpression, and proteins with stable as well as with lineage specific expression were identified. Finally, the value of antibody-based, bioimaging proteomics as a platform for biomarker discovery is demonstrated. The identification and in depth study of a candidate biomarker for colorectal cancer, SATB2, is described using both IHC and IF bioimaging. Results from extended analyses of tumor biopsies showed that detection of SATB2 protein using IHC provides a clinically relevant diagnostic tool with high specificity and sensitivity to aid in diagnosis of colorectal cancer. Furthermore, the study demonstrated a potential prognostic role of SATB2, as decreased expression was associated with a significantly shorter overall survival in patients with advanced colorectal cancer.
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| 7. |
- Lundberg, Emma, et al.
(author)
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Selection and characterization of Affibody (R) ligands to the transcription factor c-Jun
- 2009
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In: Biotechnology and applied biochemistry. - : Wiley. - 0885-4513 .- 1470-8744. ; 52, s. 17-27
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Journal article (peer-reviewed)abstract
- c-Jun is a highly oncogenic transcription factor involved in the development of different types of cancer. In the present study we have generated c-Jun-binding-affinity proteins from a phage-displayed library of so-called 'Affibody (R) ligands', developed by combinatorial engineering of a non-immunoglobulin-based scaffold protein. Homodimeric c-Jun protein was recombinantly produced in Escherichia coli and, prior to selection, the quality of the target protein was investigated by binding analyses, which indicated specific binding to a double-stranded DNA hairpin construct containing a c-Jun response element, but not to a control sequence. Isolated Affibody (R) variants from the phage selection were expressed in E. coli, purified by affinity chromatography and their interaction with c-Jun was analysed. In biosensor analyses, one Affibody (R) ligand, denoted Z(cJun518) was shown to interact with immobilized c-Jun protein with an apparent dissociation constant of 5 mu M. By constructing a head-to-tail homodimeric version of Z(cJun518), its apparent affinity for c-Jun could be increased threefold, suggesting co-operativity effects in the binding to the immobilized c-Jun protein. Further characterization of the Z(cJun518) Affibody (R) molecule demonstrated, in both affinity-capture and Western-blotting experiments, its ability to interact selectively with c-Jun, even when the c-Jun target was present in a complex protein background consisting of a bacterial cell lysate. Z(cJun518) could also be used to stain the c-Jun-overexpressing cell line C8161 visualized by confocal fluorescence microscopy. Results from competition experiments indicated that the binding epitope on c-Jun for the Z(cJun518) Affibody (R) molecule was separate from the binding sites of both a polyclonal antibody raised against the unstructured N-terminal domain and a double-stranded DNA hairpin containing a c-Jun response element. The potential intracellular use of Affibody (R) ligands directed against transcription factors and other oncogenic factors is discussed.
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| 8. |
- Lundberg, Emma, et al.
(author)
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Site-specifically conjugated anti-HER2 Affibody® molecules as one-step reagents for target expression analyses on cells and xenograft samples
- 2007
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In: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 319:1-2, s. 53-63
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Journal article (peer-reviewed)abstract
- Affibody (R) molecules are a class of small and robust affinity proteins that can be generated to interact with a variety of antigens, thus having the potential to provide useful tools for biotechnological research and diagnostic applications. In this study, we have investigated Affibody-based reagents interacting specifically with the tyrosine kinase receptor HER2. A head-to-tail dimeric construct was site-specifically conjugated with different fluorescent and enzymatic groups resulting in reagents that were used for detection and quantification. The amount of cell surface expressed HER2 oil eleven (11) well characterized cell lines was quantified relative to each other by flow cytometry and shown to correlate well with results from parallel analyses of HER2 mRNA levels measured by real-time PCR. Further, immunofluorescence I microscopy studies of the cell lines and immunohistochemical analyses of cryosections of HER2 expressing SKOV-3 xenografts showed strong staining of the plasma membrane of tumor cells with little background staining. Full-length HER2 protein Could also be efficiently recovered from a cell extract by an immunoprecipitation procedure, using an Affibody ligand-based resin. These novel non-IgG derived reagents could be used to detect and quantify HER2 expression. By adapting the methods for use with Affibody molecules binding to other cell surface receptors, it is anticipated that also these receptors can be detected and quantified in a similar manner.
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| 9. |
- Lundberg, Emma, et al.
(author)
-
The correlation between cellular size and protein expression levels--normalization for global protein profiling
- 2008
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In: Journal of proteomics. - : Elsevier BV. - 1874-3919. ; 71:4, s. 448-460
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Journal article (peer-reviewed)abstract
- An automated image analysis system was used for protein quantification of 1862 human proteins in 47 cancer cell lines and 12 clinical cell samples using cell microarrays and immunohistochemistry. The analysis suggests that most proteins are expressed in a cell size dependent manner, and that normalization is required for comparative protein quantification in order to correct for the inherent bias of cell size and systematic ambiguities associated with immunohistochemistry. Two reference standards were evaluated, and normalized protein expression values were found to allow for protein profiling across a panel of morphologically diverse cells, revealing putative patterns of over- and underexpression. Using this approach, proteins with stable expression as well as cell-line specific expression were identified. The results demonstrate the value of large-scale, automated proteome analysis using immunohistochemistry, in revealing functional correlations and establishing methods to interpret and mine proteomic data.
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| 10. |
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| 11. |
- Newberg, J. Y., et al.
(author)
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Automated analysis of human protein atlas immunofluorescence images
- 2009
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In: Proceedings - 2009 IEEE International Symposium on Biomedical Imaging. - Boston : IEEE. - 9781424439324 ; , s. 1023-1026, s. 1023-1026
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Conference paper (peer-reviewed)abstract
- The Human Protein Atlas is a rich source of location proteomics data. In this work, we present an automated approach for processing and classifying major subcellular patterns in the Atlas images. We demonstrate that two different classification frameworks (support vector machine and random forest) are effective at determining subcellular locations; we can analyze over 3500 Atlas images with a high degree of accuracy, up to 87.5% for all of the samples and 98.5% when only considering samples in whose classification assignments we are most confident. Moreover, the features obtained in both of these frameworks are observed to be highly consistent and generalizable. Additionally, we observe that the features relating the proteins to cell markers are especially important in automated learning approaches.
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| 12. |
- Palmqvist, Py, et al.
(author)
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Inhibition of hormone and cytokine-stimulated osteoclastogenesis and bone resorption by interleukin-4 and interleukin-13 is associated with increased osteoprotegerin and decreased RANKL and RANK in a STAT6-dependent pathway.
- 2006
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In: The Journal of biological chemistry. - 0021-9258 .- 1083-351X. ; 281:5, s. 2414-29
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Journal article (peer-reviewed)abstract
- Interleukin (IL)-4 and IL-13 are cytokines that inhibit bone resorption. Data showing an inhibitory effect of IL-4 and IL-13 on RANK mRNA in mouse calvariae were first reported at the 22nd American Society for Bone and Mineral Research Meeting (Lerner, U.H., and Conaway, H. H. 2000) J. Bone Min. Res. 15, Suppl. 1, Abstr. SU 230). In the present study, release of 45Ca from cultured mouse calvarial bones stimulated by different cytokines, peptides, and steroid hormones was inhibited by IL-4 and IL-13. IL-4 and IL-13 decreased receptor activator of nuclear factor-kappaB ligand (RANKL) and RANK mRNA and increased osteoprotegerin (OPG) mRNA in calvariae. Additionally, the cytokines decreased RANKL protein and increased OPG protein in calvarial bones. In osteoblasts isolated from calvariae, both an increase in RANKL mRNA and a decrease in OPG mRNA and protein elicited by vitamin D3 were reversed by IL-4 and IL-13. IL-4 and IL-13 decreased the number of tartrate-resistant acid phosphatase positive multinucleated cells and the mRNA expression of calcitonin receptor, tartrate-resistant acid phosphatase, and cathepsin K in mouse spleen cells and bone marrow macrophages (BMM) treated with macrophage colony-stimulating factor and RANKL. Inhibition of mRNA for RANK and the transcription factor NFAT2 was also noted in spleen cell and BMM cultures treated with IL-4 and IL-13. In addition, RANK mRNA and RANK protein were decreased by IL-4 and IL-13 in RAW 264.7 cells. Osteoblasts, spleen cells, and BMM expressed mRNA for the four proteins making up the IL-4 and IL-13 receptors. No effects by IL-4 on bone resorption and osteoclast formation or on RANKL and RANK mRNA expression were seen in Stat6-/- mice. The data indicate that IL-4 and IL-13, via a STAT6-dependent pathway, inhibit osteoclast differentiation and bone resorption by activating receptors on osteoblasts and osteoclasts that affect the RANKL/RANK/OPG system.
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| 13. |
- Pontén, Fredrik, et al.
(author)
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A global view of protein expression in human cells, tissues, and organs
- 2009
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In: Molecular Systems Biology. - : EMBO. - 1744-4292 .- 1744-4292. ; 5
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Journal article (peer-reviewed)abstract
- Defining the protein profiles of tissues and organs is critical to understanding the unique characteristics of the various cell types in the human body. In this study, we report on an anatomically comprehensive analysis of 4842 protein profiles in 48 human tissues and 45 human cell lines. A detailed analysis of over 2 million manually annotated, high-resolution, immunohistochemistry- based images showed a high fraction (>65%) of expressed proteins in most cells and tissues, with very few proteins (<2%) detected in any single cell type. Similarly, confocal microscopy in three human cell lines detected expression of more than 70% of the analyzed proteins. Despite this ubiquitous expression, hierarchical clustering analysis, based on global protein expression patterns, shows that the analyzed cells can be still subdivided into groups according to the current concepts of histology and cellular differentiation. This study suggests that tissue specificity is achieved by precise regulation of protein levels in space and time, and that different tissues in the body acquire their unique characteristics by controlling not which proteins are expressed but how much of each is produced. Molecular Systems Biology 5: 337; published online 22 December 2009; doi:10.1038/msb.2009.93
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| 14. |
- Stromberg, Sara, et al.
(author)
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Selective Expression of Syntaxin-7 Protein in Benign Melanocytes and Malignant Melanoma
- 2009
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In: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 8:4, s. 1639-1646
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Journal article (peer-reviewed)abstract
- To search for proteins expressed in human melanocytes and melanoma, we employed an antibody-based proteomics strategy to screen for protein expression in tissue microarrays containing normal tissues, cancer tissues and cell lines. Syntaxin-7 (STX7) was identified as a novel protein, not previously characterized in cells of melanocytic lineage, displaying a cell type-specific protein expression pattern. In tumor tissues, STX7 was expressed in malignant melanoma and lymphoma. The protein was further characterized regarding subcellular localization, specificity, tissue distribution pattern and potential as a diagnostic and prognostic marker using cell lines and tissue microarrays containing normal skin, melanocytic nevi and primary and metastatic melanoma. STX7 was expressed in normal melanocytes, various benign melanocytic nevi, atypical nevi and malignant melanoma. Analysis in two independent melanoma cohorts demonstrated STX7 expression in nearly all investigated tumors, although at varying levels (>90% positive tumors). The expression level of STX7 protein was inversely correlated to tumor stage, suggesting that decreased expression of STX7 is associated with more aggressive tumors. In conclusion, we present protein profiling data for a novel protein showing high sensitivity and specificity for cells of the melanocytic lineage. The presented antibody-based proteomics approach can be used as an effective strategy to identify novel tumor markers and evaluate their potential clinical relevance.
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| 15. |
- Vernet, Erik, et al.
(author)
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Affibody-mediated retention of the epidermal growth factor receptor in the secretory compartments leads to inhibition of phosphorylation in the kinase domain
- 2009
-
In: New biotechnology. - : Elsevier BV. - 1871-6784. ; 25:6, s. 417-423
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Journal article (peer-reviewed)abstract
- Abnormal activity of the epidermal growth factor receptor (EGFR) is associated with various cancer-related processes and motivates the search for strategies that can selectively block EGFR signalling. In this study, functional knockdown of EGFR was achieved through expression of an affibody construct, (Z(EGFR:1907))(2)-KDEL, with high affinity for EGFR and extended with the amino acids KDEL to make it resident in the secretory compartments. Expression of (Z(EGFR:1907))(2)-KDEL resulted in 80% reduction of the cell surface level of EGFR, and fluorescent staining for EGFR and the (Z(EGFR:1907))(2)-KDEL construct showed overlapping intracellular localisation. Immunocapture of EGFR from cell lysates showed that an intracellular complex between EGFR and the affibody construct had been formed, further indicating a specific interaction between the affibody construct and EGFR. Surface depletion of EGFR led to a dramatic decrease in the amount of kinase domain phosphorylated EGFR, coincident with a significant decrease in the proliferation rate.
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| 16. |
- Vernet, Erik, et al.
(author)
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Affinity-based entrapment of the HER2 receptor in the endoplasmic reticulum using an affibody molecule
- 2008
-
In: Journal of immunological methods. - : Elsevier BV. - 0022-1759. ; 338, s. 1-6
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Journal article (peer-reviewed)abstract
- Interference with the export of cell surface receptors can be performed through co-expression of specific affinity molecules designed for entrapment in the endoplasmic reticulum during the export process. We describe the investigation of a small (6 kDa) non-immunoglobulin-based HER2 receptor binding affibody molecule (ZHER2:00477), for use in affinity mediated entrapment of the HER2 receptor in the ER. Constructs encoding ZHER2:00477 or a control affibody protein, with or without ER-retention peptide extensions (KDEL), were expressed in the HER2 over-expressing cell line SKOV-3. Intracellular expression of the full-length affibody constructs could be confirmed by probing cell extracts by Western blotting. Confocal immunofluorescence microscopy experiments showed extensive co-localization of the HER2 receptor and ZHER2:00477-KDEL in the ER, whereas the use of a KDEL-extended control affibody molecule resulted in distinct and separate signals from cell surface-localized HER2 receptor and ER-localized affibody protein. This indicated a capability of the ZHER2:00477-KDEL fusion protein to functionally interfere with the export process of HER2 receptor in a specific manner. Using flow cytometry and cell proliferation analyses, it could be shown that expression of the ZHER2:00477-KDEL fusion construct in the SKOV-3 cell line resulted both in a marked reduction in cell surface level of HER2 receptors and that the cell population doubling time was significantly increased. Expression of the ZHER2:00477-KDEL fusion protein in additional cell lines of different origin and with different expression levels of endogenous HER2 receptor compared to SKOV-3, also resulted in depletion of the cell surface levels of HER2 receptor. This indicated upon a general ability of the ZHER2:00477-KDEL fusion protein to functionally interfere with the export process of HER2.
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| 17. |
- Ådahl, Emma, et al.
(author)
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From climate change to population change: the need to consider annual life cycles
- 2006
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In: Global Change Biology. - : Wiley. - 1354-1013. ; 12:9, s. 1627-1633
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Journal article (peer-reviewed)abstract
- Detailed studies of organisms' life cycles are important for understanding population response to climate change. However, in general one cannot make strong inference about the overall population response from such studies, unless the full annual cycle of the species in question is covered. Here, we present a theoretical framework for the understanding of population response to climate change. Owing to the combined effects of demography, intraspecific feedback, and a possible use of environmental cues, environmentally induced changes in survival and/or reproduction do not necessarily lead to a straightforward change in population size. This framework can guide our thinking about how abiotic conditions work their way to the population level. More specifically, it can help us to identify mechanisms that need to be examined when predicting population change in response to expected climate change.
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