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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100039	1/1	Cricetulus griseus	NAY	(d)(U)C DG	Fernández-Messina L	2010	11%
Study summary Full title Differential mechanisms of shedding of the glycosylphosphatidylinositol (GPI)-anchored NKG2D ligands All authors Fernández-Messina L, Ashiru O, Boutet P, Agüera-González S, Skepper JN, Reyburn HT, Valés-Gómez M Journal J Biol Chem Abstract Tumor cells release NKG2D ligands to evade NKG2D-mediated immune surveillance. The purpose of our in (show more...)Tumor cells release NKG2D ligands to evade NKG2D-mediated immune surveillance. The purpose of our investigation was to explore the cellular mechanisms of release used by various members of the ULBP family. Using biochemical and cellular approaches in both transfectant systems and tumor cell lines, this paper shows that ULBP1, ULBP2, and ULBP3 are released from cells with different kinetics and by distinct mechanisms. Whereas ULBP2 is mainly shed by metalloproteases, ULBP3 is abundantly released as part of membrane vesicles known as exosomes. Interestingly, exosomal ULBP3 protein is much more potent for down-modulation of the NKG2D receptor than soluble ULBP2 protein. This is the first report showing functionally relevant differences in the biochemistry of the three members of the ULBP family and confirms that in depth study of the biochemical features of individual NKG2D ligands will be necessary to understand and manipulate the biology of these proteins for therapy. (hide) EV-METRIC 11% (30th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Cricetulus griseus Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 120 Density gradient Only used for validation of main results Yes Lowest density fraction 5 Highest density fraction 60 Orientation Bottom-up Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV100039
species	Cricetulus griseus
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	11