Cd63 h5c6

A minimum of 1.0 × 10¹⁰ vesicles was used for western blot. Crude milk EVs were digested with RIPA buffer (50 mM Tris‐HCl, pH 7.5, 150 mM NaCl, 1% v/v Nonidet P‐40, 0.5% v/v sodium deoxycholate, and 0.1% SDS) supplemented with protease inhibitor cocktail (Sigma‐Aldrich, St. Louis, Missouri, USA). After heating for 5 min by 95°C, pEV lysates were centrifuged for 20 min at 12,000 × g, and supernatant was collected. Samples were loaded on a 10–12% SDS‐PAGE gel and was subjected to electrophoresis at 80 V for 30 min, thereafter 1 h at 100 V at 4°C. Proteins were blotted onto nitrocellulose membranes (on ice, 275 mA, 120 min) and blots were blocked in 5% BSA (>95% purity). Overnight incubation at 4°C with primary antibody was followed by 1 h incubation at room temperature with an HRP‐secondary antibody. Blots were visualized using enhanced chemiluminescence on the ImageQuant LAS400. Antibodies used; CD81 (B‐11), Santa Cruz, sc‐166029; HSP70 (3A3), Santa Cruz, sc‐32239: CD63 (H5C6), BD Pharmingen, 556019; ALIX (1A12), Santa Cruz, sc‐53540.

Standard western blot analysis was performed by denaturing the EV samples isolated from 250 μl of ascites and loading them onto 4–12% NuPAGE Bis-Tris Gels in reducing conditions (Thermo Fisher Scientific, USA). After gel electrophoresis, the proteins were transferred to a nitrocellulose membrane. The membrane was then probed with a primary antibody followed by a HRP-conjugated secondary antibody. The primary antibody was detected using a chemiluminescent HRP-substrate (Super Signal West Solution, Thermo Fisher Scientific, USA). The chemiluminescent signals were analyzed with Image Lab software (Bio-Rad Laboratories, USA). The band intensities of each individually analyzed protein were quantified by subtracting the background signal and normalizing to the corresponding β-actin band intensity. Results were given as the protein to β-actin ratio. The primary antibodies were mouse antibodies against the human proteins β-actin (C4), β-catenin (E-5), CD71 (H68.4), CD9 (C-4), Alix (1A12), CD59 (H-7), EpCAM (0.N.276) (Santa Cruz Biotechnology Inc., USA), CD63 (H5C6) (BD Biosciences, USA), and FN1-EDA (IST-9) (abcam PLC, UK). All primary antibodies were used in a 1 : 50 dilution; only anti-FN1-EDA was diluted 1 : 500. The secondary antibody was a goat anti-mouse IgG-HRP conjugate (sc-2031, Santa Cruz Biotechnology Inc., USA) and diluted 1 : 1250.