Alix antibody (cat. no. ab117600, Abcam), α-actinin antibody (H-2, cat. no. sc-17829, Santa-Cruz), β-actin antibody (Clone AC-74, cat. no. A2228, Sigma-Aldrich), Calnexin antibody (Clone C5C9, cat. no. 2679, Cell Signaling), CD63 antibody (cat. no. 25682–1-AP, Proteintech), HSP90α/β (F-8, cat. no. sc-13119, Santa-Cruz), TSG101 antibody (Clone 4A10, cat. no. MA1-23296, Thermo Fisher).
Western blots were used to examine the presence of common exosomal proteins in cellular fractions and sucrose gradient purified exosomes. Using Capan-2 cells as a representative example, equivalent micrograms of proteins from ER and mitochondrial (P2), cytoplasmic (S2), media (M), and exosome (Ex) fractions, prepared from different steps during exosome isolation as described above in section 1.1, were separated by SDS-PAGE and transferred to nitrocellulose membranes. Development was performed using Pierce ECL 2 Western blotting substrate (Thermo Fisher Scientific) and radiographic films (Lightlab).
Western blots were used to examine the presence of common exosomal proteins in cellular fractions and sucrose gradient purified exosomes. Using Capan-2 cells as a representative example, equivalent micrograms of proteins from ER and mitochondrial (P2), cytoplasmic (S2), media (M), and exosome (Ex) fractions, prepared from different steps during exosome isolation as described above in section 1.1, were separated by SDS-PAGE and transferred to nitrocellulose membranes. Development was performed using Pierce ECL 2 Western blotting substrate (Thermo Fisher Scientific) and radiographic films (Lightlab).