Anti cd63 h5c6

Samples were lysed with RIPA buffer (Pierce, Thermo Scientific) supplemented with a protease inhibitor mixture (Sigma-Aldrich). Protein concentration was determined with microBCA protein assay following the manufacturer’s recommendations (Pierce, Thermo Scientific). For SDS-PAGE, samples were prepared in Laemmli buffer (Bio-Rad, Hercules, CA, USA) under non-reducing conditions, and 25 μg of samples were loaded in 10–12% Mini-PROTEAN TGX™ gels (Bio-Rad) and transferred onto Protran nitrocellulose membrane. Membranes were blocked with 5% blotting-grade non-fat dry milk (Bio-Rad) in Tris-buffered saline (TBS) for 1 h at room temperature (RT). Primary antibodies were diluted in 2.5% milk-TBS: mouse monoclonal anti-CD9 (ALB 6, 1:200) and anti-GAPDH (7B, 1:500) from Santa Cruz Biotechnology (Dallas, TX, USA), anti-CD63 (H5C6, 1:200) and anti-HSP70 (7, 1:2000) from BD Pharmingen (BD Biosciences, San Jose, CA, USA), were used for Western blotting. Membranes were washed three times with TBS-0.1% Tween 20 (TBST), and incubated for 45 min at RT with the goat anti-mouse IgG-HRP secondary antibody (Santa Cruz Biotechnology) diluted in 2.5% milk-TBST. Membranes were washed, incubated with Luminata Crescendo Western HRP Substrate (Merck Millipore, Merck KGaA, Darmstadt, Germany), and visualised on Amersham Hyperfilm ECL (GE Healthcare Ltd, Chicago, IL, USA).