| 1. |
- Babakov, V.N., et al.
(författare)
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RelA/NF-?B transcription factor associates with a-actinin-4
- 2008
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 314:5, s. 1030-1038
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Tidskriftsartikel (refereegranskat)abstract
- The NF-?B/RelA family of transcription factors regulates inducible transcription of a large number of genes in response to diverse stimuli. Little is known, however, about the location of NF-?B in the cytoplasm and the transport mechanism to the nucleus. We found that NF-?B is associated with the actin-binding protein a-actinin-4. NF-?B and a-actinin-4 co-localized along actin stress fibers and in membrane lamellae in A431 cells. After a 30-min stimulation with EGF or TNF-a, a-actinin-4 and p65 were found in the nucleus. Disruption of cytoskeleton by cytochalasin D prior to treatment with TNF-a led to increase of p65 nuclear translocation. Antibodies to p65 subunit of NF-?B co-immunoprecipitated a-actinin-4 from A431 cell lysates and nuclear extracts, but a-actinin-1 and ß-actin were not found in the precipitates. Affinity chromatography experiments displayed that p65 and p50 subunits of NF-?B can bind to matrix-bound chicken gizzard a-actinin. We suggest that the a-actinin-4 is important for the NF-?B nuclear translocation and its functions inside the nucleus. © 2007 Elsevier Inc. All rights reserved.
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| 2. |
- Khotin, M.G., et al.
(författare)
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Analysis of nuclear protein complexes comprising a-actinin-4 by 2D-electrophoresis and mass-spectrometry
- 2009
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Ingår i: Tsitologiya. - : SP MAIK Nauka/Interperiodica. - 0041-3771. ; 51:8, s. 684-690
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Tidskriftsartikel (refereegranskat)abstract
- Actin-binding protein a-actinin-4 is a member of spectrin super family. It is located in the cytoplasm and in the nucleus. However, nuclear functions of a-actinin-4 are still not clear. In this study, we analyzed composition of nuclear protein complexes associated with a-actinin-4 in A431 cells. Using 2D electrophoresis, we have determined that about 50 different proteins may be associated with nuclear a-actinin-4. Using mass-spectrometry, we analyzed major proteins of these complexes. ß-Actin, a- and ß-tubulins, ribonucleoprotein A2/B1, which regulates splicing and is associated with ß-actin, peroxiredoxin-1, which is involved in oxidative stress, and glycolytic enzyme D-3-phosphoglycerate dehydrogenase were identified by MALDI-TOF. Detection of these proteins in nuclear complexes with a-actinin-4 may suggest that a-actinin-4 is involved in transcription and splicing. Presence of a-actin in the investigated complexes was confirmed by tandem mass-spectrometry (MALDITOF-TOF). Immunoprecipitation of nuclear proteins with antibodies against a-tubulin confirmed association of a-actinin-4 with a-tubulin in the protein complex. Nuclear a-actinin-4 constitutes of 105 KDa fullsize isoform and two truncated isoforms of 65 and 75 kDa, whereas only the truncated isoform have been found in nuclear complexes with a-tubulin. These data suggest that a-actinin-4 is associated with a number of different nuclear protein complexes which may carry out different functions in the cell nucleus.
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| 3. |
- Turoverova, L.V., et al.
(författare)
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Analysis of extracellular matrix proteins produced by cultured cells
- 2009
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Ingår i: Cell and Tissue Biology. - : SP MAIK Nauka/Interperiodica. - 1990-519X .- 1990-5203. ; 3:5, s. 497-502
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Tidskriftsartikel (refereegranskat)abstract
- The extracellular matrix (ECM) is a highly organized multimolecular structure essential for the vital functions of any organism. Although much of the data of extracellular matrix components has been accumulated, the isolation of an entire set of these proteins remains a complex procedure due to the high content of fibrillar proteins and proteoglycans, which form multidomain, netlike structures. In the study presented, we developed a method for isolating ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membranes. Subsequent treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibers significantly improved the fractioning of ECM proteins. The extraction of remaining proteins from the surface of the culture plate was preformed by a buffer developed based on Laemmli probe buffer. Using this method, we isolated ECM proteins synthesized by cultured cells, and the extracted proteins were suitable for future analysis by SDS PAGE and two-dimentional electrophoresis, as well as for identifying individual proteins by mass spectrometry. This study may allow us to compare assortments of ECM proteins isolated from different sources, and elucidate impact of various proteins on structure and property of extracellular matrix of investigated cells.
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| 4. |
- Turoverova, L.V., et al.
(författare)
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Analysis of extracellular matrix proteins produced by cultured cells
- 2009
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Ingår i: Tsitologiya. - St Petersburg, Russian Federation : Sankt-Peterburgskaya Izdatel'skaya Firma Nauka. - 0041-3771. ; 51:8, s. 691-696
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular matrix (ECM) is a highly organized multimolecular structure essential for vital function of any organism. Although a lot of data on the extracellular matrix components has been accumulated, an isolation of the entire set of these proteins still remains to be a complex procedure since ECM contains fibrillar proteins and proteoglycans, which form multidomain net-like structures. In the presented study, we developed a method for isolation of ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membrane. Following treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibrils significantly improved fractioning of ECM proteins. Extraction of the remained proteins from culture plate surface was preformed using a buffer developed on the basis of Laemmli probe buffer. With this method, we isolated ECM proteins synthesized by culturing cells and suitable for a future analysis by SDS PAGE and two-dimentional electrophoresis as well as for identification of individual proteins by mass-spectrometry. This study may allow comparing protein contents of ECMs isolated from different sources, and elucidate influences of various proteins on the protein and the properties of extracellular matrix of investigated cells.
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