Western blotting was performed as described previously [9] (link). Primary antibodies against CD9, CD63, CD81, HSP90, FGFBP1, SIPA1, THBS1, TGFBI, COL6A1, RPL10, SLC2A3 (GLUT3), MYO1D, RBP1, SMOC2, GLG1, and CEMIP (Protein-Tech Group, Rosemont, IL, USA) were used.
Western blotting was performed to verify the expression of exosome marker proteins and selected exoDEPs in SW620 and SW480 exosomes. Equivalent amounts of total protein (20 μg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the antibodies were diluted as follows: anti-CD9 (1:1000), anti-CD63 (1:1000), anti-CD81 (1:2000), anti-HSP90 (1:200), anti-FGFBP1 (1:500), anti-SIPA1 (1:2000), anti-THBS1 (1:500), anti-TGFBI (1:200), anti-COL6A1 (1:200), anti-RPL10 (1:1000), anti-GLUT3 (1:500), anti-MYO1D (1:200), anti-RBP1 (1:500), anti-SMOC2 (1:500), anti-GLG1 (1:1000), and anti-CEMIP (1:1000) (Protein-Tech Group, Rosemont, IL, USA). Proteins were detected using an enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Dallas, TX, USA). Western blot signals were quantified using FluorChem E (Protein Simple, San Jose, CA, USA). All analyses were performed using western blots in at least two biological replicates.
Western blotting was performed to verify the expression of exosome marker proteins and selected exoDEPs in SW620 and SW480 exosomes. Equivalent amounts of total protein (20 μg) were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the antibodies were diluted as follows: anti-CD9 (1:1000), anti-CD63 (1:1000), anti-CD81 (1:2000), anti-HSP90 (1:200), anti-FGFBP1 (1:500), anti-SIPA1 (1:2000), anti-THBS1 (1:500), anti-TGFBI (1:200), anti-COL6A1 (1:200), anti-RPL10 (1:1000), anti-GLUT3 (1:500), anti-MYO1D (1:200), anti-RBP1 (1:500), anti-SMOC2 (1:500), anti-GLG1 (1:1000), and anti-CEMIP (1:1000) (Protein-Tech Group, Rosemont, IL, USA). Proteins were detected using an enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Dallas, TX, USA). Western blot signals were quantified using FluorChem E (Protein Simple, San Jose, CA, USA). All analyses were performed using western blots in at least two biological replicates.