2.4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis

Two micrograms of proteins in the EV fractions, prepared as described above, were separated using SDS-PAGE on SuperSep HG, 5–20% gradient gels (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Protein bands were visualized using silver staining (2D-Silver Stain Reagent II, Cosmo Bio, Tokyo, Japan). To detect specific proteins in the salivary EVs, protein bands were transferred onto polyvinylidene difluoride (PVDF) membranes (BSP0161, Pall Corporation, MA, USA) using a wet transfer method (1703930JA, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Nonspecific binding sites were blocked by incubating the membranes in 100 mM Tris-HCl (pH 7.4) and 150 mM NaCl with 5% skim milk and 1% Tween 20. The membranes were then incubated overnight at 4 °C with rabbit anti-mucin 5B (1:1,000, H-300, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), goat anti-IgA (1:1,000, A80-102A, Bethyl Laboratories, Montgomery, TX, USA), goat anti-DPP IV (1:1,000, AF1180, R&D Systems, Inc., Minneapolis, MN, USA), rabbit anti-CD9 (1:1,000, EXOAB-CD9A-1, System Biosciences, Mountain View, CA, USA), goat anti-Alix (1:1,000, Q-19, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), or mouse anti-TSG101 (1:1,000, 4A10, Abcam, Cambridge, MA, USA) primary antibodies, followed by incubation with horseradish peroxidase-labeled secondary antibodies (1:5,000; anti-goat antibodies, 811620, Thermo Fisher Scientific; anti-rabbit antibodies, W4011, and anti-mouse antibodies, W4021, Promega Corporation, WI, USA). The protein bands were visualized using a LAS-4000 mini luminescent image analyzer (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA) and the ECL prime western blotting detection kit (GE Healthcare). Immunoreactive bands were quantified using the Image J software (National Institutes of Health, Bethesda, MD, USA).