Anti aip1 alix

Proteins (20 μg) were separated using bis-Tris–HCl-buffered 4%–12% gradient polyacrylamide gels (Invitrogen, Carlsbad, CA, USA) and blotted onto PVDF membranes (Millipore, Watford, UK). Membranes were blocked with 3% skim milk in TBS-T (20 mM Tris, 0.1% Tween 20, and 137 mM NaCl) at room temperature for 1 h. Primary antibodies were incubated overnight at 4°C as follows: anti-AIP1/Alix (1:250 dilution; BD Biosciences, San Jose, CA, USA), anti-CD63 (1:500 dilution; System Bioscience, Mountain View, CA, USA), anti-CD9 (1:500 dilution; System Bioscience, Mountain View, CA, USA), and anti-calnexin (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were then incubated with secondary antibodies at room temperature for 1 h after washing three times with TBS-T. Secondary antibodies were used as follows: anti-mouse for AIP1/Alix (1:2,000 dilution; Invitrogen-Molecular Probes, Eugene, Oregon), anti-rabbit for CD63 and CD9 (1:2,000 dilution; System Bioscience, Mountain View, CA, USA) and anti-goat for calnexin (1:2,000 dilution; Invitrogen-Molecular Probes, Eugene, Oregon). Immuno-reactive bands were visualized using ECL reagents (Roche, Nutley, NJ, USA) and imaged using the LAS-3000 imaging system (Fuji Film, Tokyo, Japan).