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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110003	1/1	Homo sapiens Mus musculus	NAY	(d)(U)C DG	Valapala M	2011	67%
Study summary Full title Lipid raft endocytosis and exosomal transport facilitate extracellular trafficking of annexin A2 All authors Valapala M, Vishwanatha JK Journal J Biol Chem Abstract Annexin A2 (AnxA2), a Ca(2+)-dependent phospholipid-binding protein, is known to associate with the (show more...)Annexin A2 (AnxA2), a Ca(2+)-dependent phospholipid-binding protein, is known to associate with the plasma membrane and the endosomal system. Within the plasma membrane, AnxA2 associates in a Ca(2+) dependent manner with cholesterol-rich lipid raft microdomains. Here, we show that the association of AnxA2 with the lipid rafts is influenced not only by intracellular levels of Ca(2+) but also by N-terminal phosphorylation at tyrosine 23. Binding of AnxA2 to the lipid rafts is followed by the transport along the endocytic pathway to be associated with the intralumenal vesicles of the multivesicular endosomes. AnxA2-containing multivesicular endosomes fuse directly with the plasma membrane resulting in the release of the intralumenal vesicles into the extracellular environment, which facilitates the exogenous transfer of AnxA2 from one cell to another. Treatment with Ca(2+) ionophore triggers the association of AnxA2 with the specialized microdomains in the exosomal membrane that possess raft-like characteristics. Phosphorylation at Tyr-23 is also important for the localization of AnxA2 to the exosomal membranes. These results suggest that AnxA2 is trafficked from the plasma membrane rafts and is selectively incorporated into the lumenal membranes of the endosomes to escape the endosomal degradation pathway. The Ca(2+)-dependent exosomal transport constitutes a novel pathway of extracellular transport of AnxA2. (hide) EV-METRIC 67% (94th 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 exosomes 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 Adj. k-factor 226 (pelleting) / 226 (washing) Protein markers EV: Tf-receptor/ CD81/ CD63/ LAMP1/ HSP70 non-EV: Annexin2/ Cell organelle protein Proteomics no EV density (g/ml) 1.08-1.16 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens / Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 900 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 226 Wash: Rotor Type SW28 Wash: adjusted k-factor 226 Density gradient Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ HSP70/ LAMP1/ Tf-receptor Detected contaminants Cell organelle protein/ Annexin2 ELISA Antibody details provided? No Detected EV-associated proteins LAMP1/ Tf-receptor Characterization: Particle analysis EM EM-type immune EM Image type Close-up, Wide-field

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EV-TRACK ID	EV110003
species	Homo sapiens Mus musculus
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	67