Hrp conjugated goat anti mouse

Cells and exosomes were lysed using lysis buffer. Protein lysates of exosomes (5 μg) were run on 4–20% Mini-PROTEIN TGX gel (Bio-Rad, Hercules, California, USA) and transferred to PVDF membrane. The blots were incubated separately either with mouse monoclonal anti-human CD63 antibody (Cat# ab8219, Abcam, Cambridge, MA, USA) at a dilution of 1:10000, mouse monoclonal anti-human CD81 (Cat# NB100–65805, Novus Biologicals, Littleton, CO, USA) at a dilution of 15:10000, rabbit polyclonal anti-human LAMP2B (Cat#ab18529, Abcam, Cambridge, MA, USA) at a dilution of 1:5000, rabbit polyclonal anti-human Grp94 (Cat# 2104P, Cell Signaling Technology, Danvers, MA, USA) at a dilution of 5:10000, or with mouse monoclonal anti-human β-actin (Cat# ab8224, Abcam, Cambridge, MA, USA) at a dilution of 2.5:10000 (in TBS buffer containing 2% BSA) at room temperature for 1 h followed by washing with TBS buffer. The blots were incubated with secondary antibody (horseradish peroxidase, HRP-conjugated goat anti-mouse (Cat# W402B) or goat anti-rabbit IgG (W401B) from Promega, Madison, WI, USA) at a dilution of 1:10000 (2% Milk containing TBS buffer) for 1 h at room temperature. The blots were treated with the ECL kit according to the user manual, developed on X-ray film and finally observed using Konica Minolta SRX-101A Medical Film Processor (Konica Minolta Medical & Graphic Inc., (Shanghai, China).