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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210411	1/2	Rattus norvegicus	PC12	ExoQuick	Grindheim AK	2016	25%
Study summary Full title Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2. All authors Grindheim AK, Hollås H, Raddum AM, Saraste J, Vedeler A Journal J Cell Sci Abstract Annexin A2 (AnxA2) is a multi-functional and -compartmental protein whose subcellular localisation a (show more...)Annexin A2 (AnxA2) is a multi-functional and -compartmental protein whose subcellular localisation and functions are tightly regulated by its post-translational modifications. AnxA2 and its Tyr23-phosphorylated form (pTyr23AnxA2) are involved in malignant cell transformation, metastasis and angiogenesis. Here, we show that H2O2 exerts rapid, simultaneous and opposite effects on the Tyr23 phosphorylation status of AnxA2 in two distinct compartments of rat pheochromocytoma (PC12) cells. Reactive oxygen species induce dephosphorylation of pTyr23AnxA2 located in the PML bodies of the nucleus, whereas AnxA2 associated with F-actin at the cell cortex is Tyr23 phosphorylated. The H2O2-induced responses in both compartments are transient and the pTyr23AnxA2 accumulating at the cell cortex is subsequently incorporated into vesicles and then released to the extracellular space. Blocking nuclear export by leptomycin B does not affect the nuclear pool of pTyr23AnxA2, but increases the amount of total AnxA2 in this compartment, indicating that the protein might have several functions in the nucleus. These results suggest that Tyr23 phosphorylation can regulate the function of AnxA2 at distinct subcellular sites. (hide) EV-METRIC 25% (63rd 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 Control condition Focus vesicles extracellular vesicle 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 Commercial method Protein markers EV: TSG101/ CD63/ T-cadherin/ PTyr23AnxA2/ AnxA2 non-EV: None Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells PC12 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins TSG101 Not detected EV-associated proteins T-cadherin/ PTyr23AnxA2/ AnxA2/ CD63 Characterization: Lipid analysis No

	EV210411	2/2	Rattus norvegicus	PC12	ExoQuick	Grindheim AK	2016	25%
Study summary Full title Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2. All authors Grindheim AK, Hollås H, Raddum AM, Saraste J, Vedeler A Journal J Cell Sci Abstract Annexin A2 (AnxA2) is a multi-functional and -compartmental protein whose subcellular localisation a (show more...)Annexin A2 (AnxA2) is a multi-functional and -compartmental protein whose subcellular localisation and functions are tightly regulated by its post-translational modifications. AnxA2 and its Tyr23-phosphorylated form (pTyr23AnxA2) are involved in malignant cell transformation, metastasis and angiogenesis. Here, we show that H2O2 exerts rapid, simultaneous and opposite effects on the Tyr23 phosphorylation status of AnxA2 in two distinct compartments of rat pheochromocytoma (PC12) cells. Reactive oxygen species induce dephosphorylation of pTyr23AnxA2 located in the PML bodies of the nucleus, whereas AnxA2 associated with F-actin at the cell cortex is Tyr23 phosphorylated. The H2O2-induced responses in both compartments are transient and the pTyr23AnxA2 accumulating at the cell cortex is subsequently incorporated into vesicles and then released to the extracellular space. Blocking nuclear export by leptomycin B does not affect the nuclear pool of pTyr23AnxA2, but increases the amount of total AnxA2 in this compartment, indicating that the protein might have several functions in the nucleus. These results suggest that Tyr23 phosphorylation can regulate the function of AnxA2 at distinct subcellular sites. (hide) EV-METRIC 25% (63rd 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 H2O2 treatment Focus vesicles extracellular vesicle 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 Commercial method Protein markers EV: TSG101/ PTyr23AnxA2/ AnxA2/ T-cadherin/ CD63/ pTyr23AnxA2/ Ubiquitin non-EV: None Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells PC12 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Detected EV-associated proteins CD63/ PTyr23AnxA2/ AnxA2/ TSG101 Not detected EV-associated proteins T-cadherin Detected EV-associated proteins pTyr23AnxA2/ Ubiquitin Characterization: Lipid analysis No

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EV-TRACK ID	EV210411
species	Rattus norvegicus
sample type	Cell culture
cell type	PC12
condition	Control condition	H2O2 treatment
separation protocol	ExoQuick	ExoQuick
Exp. nr.	1	2
EV-METRIC %	25	25