Anti calnexin

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).