Anti tsg101

Western blot analysis was carried out using standard methods. Briefly, cells and EV pellets were lysed on ice with lysis buffer (150 mmol/L NaCl, 50 mmol/L TRIS pH 7.4, 0.25% NP40, 1mM EDTA, 0.1% Triton X100, 0.1% SDS, 0.1 mg/mL phenylmethylsulfonyl fluoride, protease cocktail inhibitor (Merck Life Science, Milan, Italy; cat. no.04693159001), 50 mmol/L NaF, and 10 mmol/L Na₃O₄V. For Western blots, equal protein concentrations (quantified with the Bicinchoninic Acid (BCA) method, Merck Life Science, Milan, Italy; cat. no. B9643) were resolved via 12% SDS–PAGE and transferred to PVDF membranes (Biorad Laboratories S.r.L. Rome, Italy). Antibodies used were: anti-ALIX (Covalab, Bron, France; cat. no. PAB 0204, 1:1000); anti-TSG101 (Gene Tex, Irvine, CA, USA; cat. no. GTX70255); anti-calnexin (Enzo, Farmingdale, NY, USA; cat. no. ADI-SPA-860-F 1:1000); anti-GFP (Merck Millipore, Milan, Italy; cat. no. 3580, 1:1000); goat anti-rabbit IgG, HRP (Abcam, Cambridge, UK; cat. no. 6721) or goat anti-mouse IgG, HRP (Abcam, Cambridge, UK; cat. no. 6728), 1:5000. ECL Western blotting substrate (GeneTex, Irvine, CA, USA; Trident fento Western HRP substrate, cat. no. GTX14698) was added before detection with a BioRad Chemidoc XRF+ (Biorad Laboratories S.r.L. Rome, Italy). Full blots are shown in Figure S6.

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.