The tagged receptors were expressed in CHO cells which do not express the amphotropic receptor naturally. Zurück zum Zitat Wirth M, Bode J, Zettlmeisl G, Hauser H: Isolation of overproducing recombinant mammalian cell lines by a fast and simple selection procedure. Safety Consideration ; Remember that you will be working with samples containing infectious virus. Enzymatic cholesterol determination revealed that approximately 60% of the cholesterol was removed from the plasma membrane upon MBCD treatment (data not shown). Sci. NIH3T3 cells were washed with PBS, overlaid with 4°C cold 0.5% Triton X-100 in the presence of a protease inhibitor cocktail (Pefabloc, Sigma) and gently shaked at 4°C for 1 min. In summary, these data imply that a large fraction of A-MLV Env is localized in DRMs. For co-localization studies the cells were blocked a second time after incubation with the secondary antibody and stained for GM1 with FITC-conjugated cholera toxin (Calbiochem, 8 μg/ml), for cholesterol with filipin (Sigma, 50 μg/ml) or cav-1 as described above. Fowler, A. K., Twardzik, D. R., Reed, C. D., Weislow, O. S. and Heilman, A. B) A-MLV Env and cav-1 co-localization monitored by fluorescence microscopy. Actin staining confirmed colocalization of cell surface receptors with intracellular actin filaments. pMLVampho contains the complete genome of A-MLV cloned into pBluescript (Genethon, France received via J.-C. Using density gradient centrifugation and immunocytochemical analyses, we show that Env co-localizes with cholesterol, ganglioside GM1 and caveolin-1 in these specific regions of the plasma membrane. We suggested that this phenomenon could be due to the involvement of DRMs in assembly and budding of A-MLV. The amounts shown are determined as percentages of the total of all dots; DRM (fractions 1 to 3), non-DRM (fractions 4 to 6). PubMedCentralCrossRefPubMed. Curr Biol 2003, 13: 686-690. J Virol 2000, 74: 3264-3272. The specific lipid composition of DRMs leads to the selective incorporation and concentration of specific cellular proteins (reviewed in [1]). All authors read and approved the final manuscript. Nussbaum, O., Roop, A. and Anderson, W. F. (1993). The secondary antibodies, rabbit anti rat and goat anti rabbit coupled to HRP, were used at a 1:1000 dilution. Below are the links to the authors’ original submitted files for images. The fractions were analysed for their egfp-N1 or A-MLV Env protein content using a 12% SDS gel and Western Blot. Manage cookies/Do not sell my data we use in the preference centre. NIH3T3 cells were seeded onto chamber slides (Nunc) and grown to 80% confluency. J. Virol. Characterization of a naturally occurring ecotropic receptor that does not facilitate entry of all ecrotropic murine retroviruses. Part of Proc Natl Acad Sci USA 1994, 91: 12130-12134. 10.1146/annurev.biochem.67.1.199 CrossRefPubMed, Zurück zum Zitat Schmid SL, Smythe E: Stage-specific assays for coated pit formation and coated vesicle budding in vitro. Proc Natl Acad Sci USA 1988, 85: 900-904. The author(s) declare that they have no competing interests. Hybridoma cell lines were used for the production of rat monoclonal immunoglobulin G (IgG) antibodies against MLV SU (83A25, kindly provided by L.H.Evans [21]). Acad. 10.1073/pnas.172196599 PubMedCentralCrossRefPubMed, Zurück zum Zitat Pickl WF, Pimentel-Muinos FX, Seed B: Lipid rafts and pseudotyping. Interactions between saturated acyl chains confer detergent resistance on lipids and glycophosphatidyinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behaviour. To investigate the association of A-MLV Env with DRMs via density gradient centrifugation, 293T cells were transiently transfected with a pHIT-derived plasmid encoding the A-MLV envelope protein [15]. Scatchard analysis performed with 125I-labeled AS208 showed curvilinear plots with downward concavity, indicating that receptor cooperativity participates in binding efficiency. Edited by: Coffin JM, Hughes SH, Varmus HE. Cookies policy. Zurück zum Zitat Nguyen DH, Hildreth JE: Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts. Cosset, F. L., Morling, F. J., Takeuchi, Y., Weiss, R. A., Collins, M. K. L. and Russel, S. J. Lipid composition and fluidity of the human immunodeficiency virus. By using this website, you agree to our Due to disruption of the DRM structure, a combined MBCD/TX-100 treatment should result in dispersal of DRMs and proteins concentrated therein. J. Virol. J. Virol. A fluorescence microscope (Axiovert TV135, Zeiss; filter sets: filipin – XF113, 387/450 nm (Em/Ex), FITC – 495/520 nm, Texas Red – 595/615 nm; Omega filters) at 1000× magnification was used for the detection of the stained proteins. Fraction 1 corresponds to the top and fraction 6 corresponds to the bottom of the tube. The cells were incubated with filipin, a cholesterol-binding fluorescent dye [20], and DRM markers GM1 and cav-1 were detected using FITC labelled cholera toxin or anti cav-1 antibody, respectively. Tearina, T. H. and Dornburg, R. (1995). Cav-1 is a major component of caveolae, which are flask-shaped invaginations of the plasma membrane involved in endocytic processes.

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