Clone 4a10

Samples were mixed with 20 mM TrisHCL 1% SDS and then sonicated for five minutes, three times, with vortexing in between. Volumes corresponding to 25 µg of protein from isolates were separated on a 10% polyacrylamide gel. Samples were then transferred onto a nitrocellulose membrane (Bio-Rad laboratories, Hercules, CA, USA) which was then blocked with 5% Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad Laboratories) in Tris-buffer saline (TBS) for two hours. Membrane was then incubated with primary antibodies against calnexin (1:1000; clone H-70; Santa Cruz Biotechnology, Santa Cruz, CA, USA), TSG101 (1:1000; clone 4A10; Abcam, Cambridge, UK) and CD81 (1:800; clone H-121; Santa Cruz Biotechnology) dissolved in 0.25% Blotting Grade Blocker Non-Fat Dry Milk in TBS-Tween (TBST) overnight at 4°C, after which the membrane was washed with TBST for 10 minutes, three times. Secondary antibodies [for calnexin and CD81: (1:10 000) ECL anti-rabbit IgG horseradish peroxidase-linked F(ab’)₂ fragment (donkey-anti-rabbit); for TSG101: (1:2000) ECL anti-mouse IgG horseradish peroxidase-linked F(ab’)₂ fragment (sheep-anti-mouse); GE Healthcare, Buckinghamshire, UK] were diluted in 0.25% Blotting Grade Blocker Non-Fat Dry Milk in TBST and incubated for 1.5 hours. Membranes were analysed with ECL Prime Western Blotting Detection (GE Healthcare) and a VersaDoc 4000 MP (Bio-Rad Laboratories).