Pierce micro bcatm protein assay

Total proteins from cells were extracted using RIPA buffer containing 7 × protease inhibitor (Roche, Catalogue No. 11836153001), and solubilized proteins were quantified using the Pierce Micro BCA^TM Protein Assay (Thermo Scientific, Rockford, IL, USA). Proteins were resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS- PAGE), at 10%, and transferred to polyvinylidene difluoride membranes (PVDF; Biorad, Mississauga, ON, Canada). The membranes were probed with indicated primary antibodies, and appropriate horseradish peroxidise-conjugated secondary anti-mouse (Biorad # 170-6516), or anti-rabbit (Cell Signaling # 7074S) antibodies. Amersham ECL Western Blotting Detection Kit (RPN2108 GE Healthcare) was used for the detection of chemiluminescence and band visualisation using ChemiDoc MP system (Biorad).

RIPA buffer containing protease inhibitor (Roche, Mississauga, ON, Canada) was used to isolate total proteins from cells or EVs. Lysates were incubated on ice for 5 min, centrifuged at 13,000 rpm for 10 min at 4°C, and solubilized proteins were quantified using the Pierce Micro BCA^TM Protein Assay (Thermo Scientific, Rockford, IL, USA). Proteins were resolved using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), at 10%, and transferred to polyvinylidene difluoride membranes (PVDF; Biorad, Mississauga, ON, Canada). The membranes were probed with indicated primary antibodies, and appropriate horseradish peroxidise (HRP)-conjugated secondary anti-mouse (Biorad # 170–6516), or anti-rabbit (Cell Signaling # 7074S) antibodies. Chemiluminescence (GE Healthcare) was visualized using ChemiDoc MP system (Biorad). Primary antibodies included: rabbit anti-CD81 (ab155760 Abcam), rabbit anti-CD9 (ab92726 Abcam), rabbit anti-CD63 (ab134045 Abcam or 556019 BD), rabbit anti-CD82 (12439 Cell signalling), rabbit anti-ANXA6 (ab31026 Abcam), rabbit anti-CAV1 (ab2910 Abcam), rabbit anti-SYN (ab133267 Abcam), mouse anti-FLOT1 (610821 BD), rabbit anti-RAB6B (ab206110 Abcam), mouse anti-RAB27A (5873-MO2 Abnova), rabbit anti-WNT11 (ab31962 Abcam), rabbit anti-WNT16 (ab109437 Abcam) and rabbit anti-ITGα5 (ab117611 Abcam).