Anti cd9 clone c 4

Total of 5*10¹⁰ particles of all samples were prepared with 6x Laemmli sample buffer with 10% β-mercaptoethanol and run on 4–20% gradient SDS-PAGE (Mini-Protean TGX Precast protein gel, Bio-Rad, Hercules, USA). For Western blot analysis, proteins were transferred to PVDF membrane (Immobilon-P, Merck Millipore, Burlington, USA) at 200 mA for 90 minutes using wet transfer. Nonspecific binding was blocked by with 3% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 hour followed by primary antibody incubation anti-TSG101 (Clone 51/TSG101 BD Biosciences, 1:500), anti-CD41 (clone SZ22, Beckman Coulter, 1:2000), or anti-CD9 (clone C-4, Santa Cruz Biotechnology, Dallas, USA, 1:500) overnight at 4˚C. The membranes were washed three times with TBST followed by incubation with horseradish peroxidase–conjugated anti-mouse secondary antibody in dilution 1:3000 (GE Healthcare Bio-Sciences AB). After washing, the signal was detected using Bio-Rad Clarity Western ECL Substrate (Bio-Rad) and imaged using LAS3000 imaging system (Fuji, Minato, Japan).