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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140141	1/1	Homo sapiens Canis familiaris	Urine	(d)(U)C DG	Chacon-Heszele MF	2014	29%
Study summary Full title The exocyst and regulatory GTPases in urinary exosomes All authors Chacon-Heszele MF, Choi SY, Zuo X, Baek JI, Ward C, Lipschutz JH Journal Physiol Rep Abstract Cilia, organelles that function as cellular antennae, are central to the pathogenesis of ciliopathie (show more...)Cilia, organelles that function as cellular antennae, are central to the pathogenesis of ciliopathies, including various forms of polycystic kidney disease (PKD). To date, however, the molecular mechanisms controlling ciliogenesis and ciliary function remain incompletely understood. A recently proposed model of cell-cell communication, called urocrine signaling, hypothesizes that a subset of membrane bound vesicles that are secreted into the urinary stream (termed exosome-like vesicles, or ELVs), carry cilia-specific proteins as cargo, interact with primary cilia, and affect downstream cellular functions. This study was undertaken to determine the role of the exocyst, a highly conserved eight-protein trafficking complex, in the secretion and/or retrieval of ELVs. We used Madin-Darby canine kidney (MDCK) cells expressing either Sec10-myc (a central component of the exocyst complex) or Smoothened-YFP (a ciliary protein found in ELVs) in experiments utilizing electron gold microscopy and live fluorescent microscopy, respectively. Additionally, human urinary exosomes were isolated via ultracentrifugation and subjected to mass-spectrometry-based proteomics analysis to determine the composition of ELVs. We found, as determined by EM, that the exocyst localizes to primary cilia, and is present in vesicles attached to the cilium. Furthermore, the entire exocyst complex, as well as most of its known regulatory GTPases, are present in human urinary ELVs. Finally, in living MDCK cells, ELVs appear to interact with primary cilia using spinning disc confocal microscopy. These data suggest that the exocyst complex, in addition to its role in ciliogenesis, is centrally involved in the secretion and/or retrieval of urinary ELVs. (hide) EV-METRIC 29% (60th 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 Urine 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 Protein markers EV: non-EV: Proteomics yes Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens / Canis familiaris Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Density gradient Lowest density fraction 5 Highest density fraction 30 Orientation Top-down Speed (g) 150000 Characterization: Protein analysis

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EV-TRACK ID	EV140141
species	Homo sapiens Canis familiaris
sample type	Urine
condition	NAY
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	29