Anti rab27

EV-containing fractions were lysed in 1X RIPA lysis buffer. Protein concentrations were determined by BCA protein assay kit (Thermo Fisher). Equivalent total protein amounts from BH and EVs were separated on 4-15% stain-free pre-cast SDS-PAGE gradient gels (Bio-Rad) under non-reducing conditions and transferred onto PVDF membranes (Sigma Aldrich). After 1 hour blocking in 5% non-fat milk solution (Bio-Rad 170-6404) at room temperature, membranes were incubated with anti-CD63 (1:1000 dilution), anti-Bip (1:1000 dilution) (BD Biosciences 556019 and 610978, respectively), anti-CD81(1:1000 dilution), anti-Rab27 (1:1000 dilution) (Santa Cruz Biotechnology sc23962, sc74586), anti-TSG101 (1:500 dilution), anti-CD9 (1:500 dilution), anti-Syntenin (1:500 dilution), anti-Calnexin (1:2000 dilution), or anti-GM130 (1:1000 dilution) (the last five antibodies were Abcam ab125011, ab92726, ab133267, ab22595, and ab76154) overnight at 4°C. The membrane was washed 3 times for 8 minutes in PBST while shaking, then incubated with HRP-conjugated secondary antibody (1:10000 dilution) (Santa Cruz Biotechnology sc-2357, sc-516102) at room temperature for 1 hour. After washing again in PBST, the enzymelinked antibody was detected by incubation with Pico chemiluminescent substrate (Thermo Fisher 34580) and recording on film (Millipore Sigma GE28-9068 -38).