2.8. Western Blot Analysis

The total cell lysates for colon cancer cells (10 µg) and exosomes (10 µg) were separated on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at 120 V for 90 min and transferred to nitrocellulose membranes. After blocking in 5% non-fat milk in tris-buffered saline with tween 20 (TBST) for 2 h, the membranes were blotted with primary antibodies at 4 °C overnight for 16 h, followed by secondary bodies at a dilution of 1:2000. The protein bands were visualized using WesternBright ECL solution (Advansta, San Jose, CA, USA) and the signals were captured using a C-digit blot scanner (LI-COR). Primary antibodies against KRAS, HSC70, CD63, CD9 and CD81, and secondary antibodies (mouse anti-rabbit and goat anti-mouse) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against actin and p53 were purchased from Millipore (Burlington, VT, USA) while primary antibodies against ACSL4 were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Western blotting was performed in triplicate for each tested protein.