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Protein concentrations were determined in both fractions using a BCA procedure (Pierce Biochemical, Rockford, IL, USA).
We then resuspended the microsomal pellet in incubation medium at a tissue concentration of 1 g/mL and determined protein concentrations of both fractions using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL).
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Each gradient was fractionated into 40 fractions using a Density Gradient Fractionation System (Brandel, Gaithershurg, MD).
HeLa cells were fractionated into cytosolic, membrane/organelles and nuclear fractions using a ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem) following the manufacturer's indications.
We then analyzed the fractions using a Q Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA).
The extracted sample was separated in three fractions using a Bond-elute column chromatography (Bond phase NH2, 500 mg, 40 μm particle size).
To verify that the identified miRs are associated directly with exosomes, we separated the mutp53-derived exosomal isolations into fractions using a sucrose density gradient technique.
The sediment was separated into allogenic and authigenic fractions using a wet chemical extraction technique.
T lymphocytes were isolated from the CD14 negative fractions using a pan T cell isolation kit.
The chamber was emptied in 8 ml fractions using a fraction collector, and fractions containing peak amounts of cells were identified using a 340 nm UV light source.
CM and NM cells were separated into fractions using a modified Langendorff apparatus.
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