Anti cd63

Cells were transfected with small interfering RNA (siRNA) using Oligofectamine (Invitrogen) on days 1 and 2 and harvested on day 4. siRNA duplexes were synthesized by Dharmacon, the target sequences were: 5′-CCA GUC UUC UCU CGU CCU A-3′ (Tsg101), 5′-AGA GAC AAG UGG AGG UAA A-3′ (Hrs), both as previously described 18 (link), 5′-GTT CTT GCT CTA CGT CCT C-3′ (CD63), as previously described 16 (link). Negative control siRNA was used for control cells (Mammalian AllStar negative siRNA, Qiagen). Efficiency of knockdown was assessed by western blotting using anti-Hrs (Enzo Lifesciences), anti-Tsg101 (Genetex) and anti-CD63 (1B5, a generous gift from Mark Marsh, University College, London) antibodies.
For concurrent depletion and expression studies, HeLa cells were transfected with siRNA on days 1 and 2 and transfected with PMEL (a generous gift from Michael Marks, University of Pennsylvania) on day 3 using Lipofectamine 2000 reagent (Invitrogen), following manufacturer's guidelines.

PCa cells and exosomal lysates were prepared using RIPA buffer (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and total proteins were quantified using bicinchoninic acid (BCA) assay reagents (Thermo-Scientific, Rockford, IL, USA). About 10–20 μg protein lysates were fractionated on 4–20% SDS-PAGE. Proteins were then transferred into nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), blocked in 5% BSA for 1h at room temperature. Membranes were incubated overnight at 4 °C with anti-integrin α2 (ITGA2), anti-CD9, anti-CD63, anti-E-cadherin, and anti-calnexin (GeneTex, Irvine, CA, USA), anti-c-Myc, anti-phosphorylated and total FAK, anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Vimentin (Millipore), anti-pERK1/2 (Cell Signaling, Danvers, MA, USA) antibodies. Membranes were incubated with appropriate secondary antibody for 1 h at room temperature. The specific protein bands were developed using the Clarity ^TM Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. The developed signals were visualized by Odyssey ^® Fc Imager and C-Digit Blot Scanner (LI-COR, Lincoln, NE, USA) and the densitometric analysis was performed by the Image studio Lite (LI-COR, Lincoln, NE, USA).