Precasted polyacrylamide gels

Cell extracts were prepared in Laemmli buffer (4% SDS, 20% glycerol, 120 mM Tris–HCl pH 6.8) supplemented with Pierce^TM reducing sample buffer (Thermo Scientific, Waltham, MA, USA). Proteins were resolved by SDS–PAGE using precasted polyacrylamide gels (Biorad, Hercules, CA, USA) and transferred to PVDF membranes (Biorad). Immunoblots were performed using the appropriate primary antibodies and the relative HPR-coupled secondary antibodies (GE Healthcare, Chicago, IL, USA). Proteins were visualized on a ChemiDoc MP imaging system (Biorad) using the ClarityTM Western ECL Blotting Substrate (Biorad). Antibodies are specified in Table 1.

For western blotting, 150k pellets were lysed in RIPA buffer (150-mM NaCl; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% SDS; 50-mM Tris pH 8.0; 1× protease inhibitor cocktail (Roche)). Protein concentration was measured using bicinchoninic acid. Loads normalised to 4-μg protein were resolved on 10% homemade polyacrylamide gels or 4–15% pre-casted polyacrylamide gels (Biorad), then transferred onto PVDF membrane and processed as described [20]. The following primary antibodies were used: anti-calnexin (1:10,000, ADI-SPA-860D Enzo Life Science), anti-CD63 (1:300, 143902 Biolegend; in non-reducing environment), anti-CD9 (1:300, EPR2949 Abcam), anti-flotilin-1 (1:200, 610821 BD Transduction) and anti-laminin-α1 (1:1,000, kind gift from T. Sasaki). Immuno-reactive bands were detected using chemiluminescence (Super Signal Chemiluminescent Substrate; Thermo Scientific) and images were acquired using Fusion Solo S (Vilber Lourmat).