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You searched for: EV120009 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
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
Experiment number
  • Experiments differ in Isolation method
Experiment number
  • Experiments differ in Isolation method
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV120009 2/2 Mus musculus Cell culture supernatant dUC
Sucrose-DG (valid.)
Putz U 2012 67%

Study summary

Full title
All authors
Putz U, Howitt J, Doan A, Goh CP, Low LH, Silke J, Tan SS
Journal
Sci Signal
Abstract
Exosomes are microvesicles of endosomal origin that are secreted, and their contents (proteins, lipi (show more...)Exosomes are microvesicles of endosomal origin that are secreted, and their contents (proteins, lipids, DNA, or microRNAs) can alter the physiological states of recipient cells. We demonstrated that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein normally localized in the cytoplasm and nucleus, was secreted in exosomes. Secreted PTEN was internalized by recipient cells with resultant functional activity, which resulted in reduced phosphorylation of the serine and threonine kinase Akt and reduced cellular proliferation. PTEN secretion in exosomes required Ndfip1, an adaptor protein for members of the Nedd4 family of E3 ubiquitin ligases. Without Ndfip1, neither Nedd4-1 nor Nedd4-2 promoted the recruitment of PTEN into exosomes. In addition, lysine 13 within PTEN, which is required for its ubiquitination by Nedd4-1, was required for exosomal transport of PTEN. These results implicate Ndfip1 as a molecular regulator of the exosomal export of PTEN, with consequences for non-cell-autonomous PTEN activity. Thus, we suggest that the ability of PTEN to exert phosphatase activity beyond the cell in which it is produced has implications for PTEN function during development, health, and disease. (hide)
EV-METRIC
67% (98th 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/ Flotilin1/ HSP90/ TSG101/ Beta-actin
non-EV: Cell organelle protein
Proteomics
no
EV density (g/ml)
1.08-1.14
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 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
Bottom-up
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Flotilin1/ HSP90/ TSG101/ Beta-actin
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Beta-actin
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120009 1/2 Homo sapiens Cell culture supernatant dUC Putz U 2012 22%

Study summary

Full title
All authors
Putz U, Howitt J, Doan A, Goh CP, Low LH, Silke J, Tan SS
Journal
Sci Signal
Abstract
Exosomes are microvesicles of endosomal origin that are secreted, and their contents (proteins, lipi (show more...)Exosomes are microvesicles of endosomal origin that are secreted, and their contents (proteins, lipids, DNA, or microRNAs) can alter the physiological states of recipient cells. We demonstrated that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein normally localized in the cytoplasm and nucleus, was secreted in exosomes. Secreted PTEN was internalized by recipient cells with resultant functional activity, which resulted in reduced phosphorylation of the serine and threonine kinase Akt and reduced cellular proliferation. PTEN secretion in exosomes required Ndfip1, an adaptor protein for members of the Nedd4 family of E3 ubiquitin ligases. Without Ndfip1, neither Nedd4-1 nor Nedd4-2 promoted the recruitment of PTEN into exosomes. In addition, lysine 13 within PTEN, which is required for its ubiquitination by Nedd4-1, was required for exosomal transport of PTEN. These results implicate Ndfip1 as a molecular regulator of the exosomal export of PTEN, with consequences for non-cell-autonomous PTEN activity. Thus, we suggest that the ability of PTEN to exert phosphatase activity beyond the cell in which it is produced has implications for PTEN function during development, health, and disease. (hide)
EV-METRIC
22% (55th 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
Protein markers
EV: Flotilin1/ Beta-actin
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ Beta-actin
ELISA
Detected EV-associated proteins
Beta-actin
Characterization: Particle analysis
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