Anti hsp90β

Equal amount of exosomal proteins (20 μg/sample) from each sample were mixed with 2× Laemmli’s buffer (to make the final concentration of 1× Laemmli’s buffer) and resolved by 12% SDS-PAGE at 150 V for approximately 2 h using SE260 mini-Vertical electrophoresis unit (GE Healthcare; Uppsala, Sweden). After the completion of SDS-PAGE, the resolved proteins were transferred onto a nitrocellulose membrane (Whatman, Dassel, Germany) using a semidry transfer apparatus (GE Healthcare) at 85 mA for 1.5 h. Non-specific bindings were blocked with 5% skim milk in PBS at 25°C for 1 h. The membrane was incubated with mouse monoclonal anti-heat shock protein 70 (anti-HSP70), anti-Rab5, anti-HSP90β, anti-vimentin, or rabbit polyclonal anti-Rab7 antibody (all were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA and were diluted 1:1,000 in 1% skim milk/PBS) at 4°C overnight. After washing with PBS three times, the membrane was incubated with corresponding secondary antibody conjugated with horseradish peroxidase (1:2,000 in 1% skim milk/PBS; DAKO Glostrup, Denmark) at 25°C for 1 h. Immunoreactive bands were developed by SuperSignal West Pico chemiluminescence substrate (Pierce Biotechnology, Rockford, IL, USA) and were then visualized by autoradiogram.