Western Blot analysis
All of the following antibodies were purchased from ThremoFisher
Scientific, USA; MFGE8 (Lactadherin) Monoclonal Antibody (EDM45), MFGE8
(Lactadherin) Polyclonal Antibody, and CD63 goat polyclonal antibody and
used for Western Blot analyses. Samples containing EV pellets were
suspend in PBS and diluted with lysis buffer, then were further mixed
with 6x SDS sample buffer and placed at 95°C for 5 min. Gel (12%
Mini-PROTEAN TGX Stain-Free, Bio-Rad, USA) run 20-25 minute at 90 V for
stacking, then 1-1.5 hour at 110 V for separating. The proteins were
transferred using Transfer-Blot Turbo Transfer System with Transfer pack
(Bio-Rad). The membranes were blocked with 5% non-fat dried milk
dissolved in TBST. The proteins were detected by incubation with
secondary antibody conjugated with HRP (anti-rabbit IgG goat antibody,
anti-rabbit IgG donkey antibody, and anti-goat IgG donkey antibody all
by Invitrogen, USA). The membranes were washed thrice prior to imaging.
Detected the peroxidase activity using a Clarity TMWestern ECL substrate (Bio-Rad) and visualized using an ChemiDocTM Imaging system (Bio-Rad).