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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV130022	1/3	Homo sapiens Mus musculus	Cell culture supernatant	dUC Sucrose-DG (valid.)	Zhu H	2013	78%
Study summary Full title Mutation of SIMPLE in Charcot-Marie-Tooth 1C alters production of exosomes All authors Zhu H, Guariglia S, Yu RY, Li W, Brancho D, Peinado H, Lyden D, Salzer J, Bennett C, Chow CW Journal Mol Biol Cell Abstract Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small inte (show more...)Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE) account for the rare autosomal-dominant demyelination in CMT1C patients. Understanding the molecular basis of CMT1C pathogenesis is impeded, in part, by perplexity about the role of SIMPLE, which is expressed in multiple cell types. Here we show that SIMPLE resides within the intraluminal vesicles of multivesicular bodies (MVBs) and inside exosomes, which are nanovesicles secreted extracellularly. Targeting of SIMPLE to exosomes is modulated by positive and negative regulatory motifs. We also find that expression of SIMPLE increases the number of exosomes and secretion of exosome proteins. We engineer a point mutation on the SIMPLE allele and generate a physiological mouse model that expresses CMT1C-mutated SIMPLE at the endogenous level. We find that CMT1C mouse primary embryonic fibroblasts show decreased number of exosomes and reduced secretion of exosome proteins, in part due to improper formation of MVBs. CMT1C patient B cells and CMT1C mouse primary Schwann cells show similar defects. Together the data indicate that SIMPLE regulates the production of exosomes by modulating the formation of MVBs. Dysregulated endosomal trafficking and changes in the landscape of exosome-mediated intercellular communications may place an overwhelming burden on the nervous system and account for CMT1C molecular pathogenesis. (hide) EV-METRIC 78% (99th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Protein markers EV: Alix/ CD63/ Flotilin1/ HSP70/ Hsc70/ SIMPLE non-EV: Tubulin Proteomics no EV density (g/ml) 1.130 TEM measurements 40-100 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens / Mus musculus Sample Type Cell culture supernatant Isolation Method Differential ultracentrifugation 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: time(min) 60 Density gradient Only used for validation of main results 1 Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Top-down Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ Flotilin1/ HSP70/ Hsc70/ SIMPLE Detected contaminants Tubulin ELISA Detected EV-associated proteins Hsc70/ SIMPLE Characterization: Particle analysis NTA EM EM-type immune EM/ scanning EM Image type Close-up, Wide-field

	EV130022	2/3	Homo sapiens	CMT1C patient B cells	dUC Sucrose-DG (valid.)	Zhu H	2013	56%
Study summary Full title Mutation of SIMPLE in Charcot-Marie-Tooth 1C alters production of exosomes All authors Zhu H, Guariglia S, Yu RY, Li W, Brancho D, Peinado H, Lyden D, Salzer J, Bennett C, Chow CW Journal Mol Biol Cell Abstract Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small inte (show more...)Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE) account for the rare autosomal-dominant demyelination in CMT1C patients. Understanding the molecular basis of CMT1C pathogenesis is impeded, in part, by perplexity about the role of SIMPLE, which is expressed in multiple cell types. Here we show that SIMPLE resides within the intraluminal vesicles of multivesicular bodies (MVBs) and inside exosomes, which are nanovesicles secreted extracellularly. Targeting of SIMPLE to exosomes is modulated by positive and negative regulatory motifs. We also find that expression of SIMPLE increases the number of exosomes and secretion of exosome proteins. We engineer a point mutation on the SIMPLE allele and generate a physiological mouse model that expresses CMT1C-mutated SIMPLE at the endogenous level. We find that CMT1C mouse primary embryonic fibroblasts show decreased number of exosomes and reduced secretion of exosome proteins, in part due to improper formation of MVBs. CMT1C patient B cells and CMT1C mouse primary Schwann cells show similar defects. Together the data indicate that SIMPLE regulates the production of exosomes by modulating the formation of MVBs. Dysregulated endosomal trafficking and changes in the landscape of exosome-mediated intercellular communications may place an overwhelming burden on the nervous system and account for CMT1C molecular pathogenesis. (hide) EV-METRIC 56% (50th 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 CMT1C patient B cells Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Protein markers EV: Alix/ CD63/ "SIMPLE/ Clathrin HC" non-EV: Tubulin Proteomics no TEM measurements >100 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type CMT1C patient B cells Isolation Method Differential ultracentrifugation 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: time(min) 60 Density gradient Only used for validation of main results 1 Lowest density fraction 0.25 Highest density fraction 2.5 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ "SIMPLE/ Clathrin HC" Detected contaminants Tubulin ELISA Detected EV-associated proteins "SIMPLE/ Clathrin HC" Characterization: Particle analysis NTA EM EM-type scanning EM Image type Wide-field

	EV130022	3/3	Mus musculus	Blood plasma	Commercial Other method	Zhu H	2013	0%
Study summary Full title Mutation of SIMPLE in Charcot-Marie-Tooth 1C alters production of exosomes All authors Zhu H, Guariglia S, Yu RY, Li W, Brancho D, Peinado H, Lyden D, Salzer J, Bennett C, Chow CW Journal Mol Biol Cell Abstract Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small inte (show more...)Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE) account for the rare autosomal-dominant demyelination in CMT1C patients. Understanding the molecular basis of CMT1C pathogenesis is impeded, in part, by perplexity about the role of SIMPLE, which is expressed in multiple cell types. Here we show that SIMPLE resides within the intraluminal vesicles of multivesicular bodies (MVBs) and inside exosomes, which are nanovesicles secreted extracellularly. Targeting of SIMPLE to exosomes is modulated by positive and negative regulatory motifs. We also find that expression of SIMPLE increases the number of exosomes and secretion of exosome proteins. We engineer a point mutation on the SIMPLE allele and generate a physiological mouse model that expresses CMT1C-mutated SIMPLE at the endogenous level. We find that CMT1C mouse primary embryonic fibroblasts show decreased number of exosomes and reduced secretion of exosome proteins, in part due to improper formation of MVBs. CMT1C patient B cells and CMT1C mouse primary Schwann cells show similar defects. Together the data indicate that SIMPLE regulates the production of exosomes by modulating the formation of MVBs. Dysregulated endosomal trafficking and changes in the landscape of exosome-mediated intercellular communications may place an overwhelming burden on the nervous system and account for CMT1C molecular pathogenesis. (hide) EV-METRIC 0% (median: 14% 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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation 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 Blood plasma Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Commercial + Other method Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Mus musculus Sample Type Blood plasma Isolation Method Other Name other isolation method Microfluidics Characterization: Particle analysis NTA

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