Tris buffered saline (tbs)

Proteins were extracted with 1% Triton X100-containing lysis buffer: 50 mM HEPES (pH: 7.5); 150 mM NaCl; 10 mM glucose; 2 units/mL hexokinase (Sigma); and protease inhibitor cocktails. Detergent-soluble and insoluble fractions were separated by centrifugation at 18,000× g at 4 °C for 10 min. Protein concentration of the soluble fraction was determined by a standard Bradford assay system. For pulldown experiments, the cell lysate was treated using anti-Flag agarose beads (Sigma), as previously described [22 (link)]. Protein samples were subjected to reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The membrane was blocked with tris-buffered saline containing 5% skim milk and 0.05% Tween 20 (Nacalai, Kyoto, Japan) at 4 °C overnight. Next, the proteins were detected using indicated primary antibodies and secondary antibodies. Finally, they were visualized with enhanced chemiluminescence and the ChemiDoc system (Bio-Rad, Hercules, CA, USA). Densitometric analysis was performed using the ImageJ software version 1.53 (National Institute of Health, Bethesda, MD, USA). The expression of proteins of interest was normalized to that of αTubulin. The error bars in figures represent the standard deviation for at least three independent experiments.

EVs were labeled with FM4-64 and the enumeration and sizing of labeled and non-labeled EVs were performed with a ViewSizer 3000 (software version 1.9.0.4518, HORIBA, Kyoto, Japan) using a 520-nm laser and a 650-nm filter. The zeta potential of EVs was assessed by a zeta potential analyzer (ELSZ-2000, Otsuka Electronics, Osaka, Japan) at 25 °C. EVs suspended in TBS (pH 7.4, Nacalai Tesque) were deposited on a carbon film-coated copper grid treated by glow discharge and then stained with 2% uranyl acetate solution for negative staining. The prepared grid was observed with the transmission electron microscope JEM-1400 (JEOL Inc., Tokyo, Japan).