Tsg101

Tissues, cells or exosomes were lysed in radioimmunoprecipitation lysis buffer (RIPA) containing PMSF (100:1 dilution), and total protein concentration was determined using a BCA protein quantification kit. The protein samples were separated through 10% SDS-containing polyacrylamide gel (SDS-PAGE), then transferred onto 0.22-μm polyvinylidene difluoride (PVDF) membranes. Next, the membranes were blocked in TBST containing 5% skimmed milk for 1.5 h. After washing with PBS three times, PVDF membranes were incubated at 4 °C overnight with antibodies against the following proteins: TSG101 (Immunoway, USA), CD9 (Immunoway), ALIX (Immunoway), PTEN (Immunoway), TOB1 (Immunoway), BCL-2 (Immunoway), BAX (Immunoway), cleaved-caspase 3 (CST, USA), E-cadherin (CST), N-cadherin (CST), vimentin (CST), or β-Actin (CST). The membranes were washed three times with PBS, then incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody for 1 h at room temperature. The protein bands were visualized by enhanced chemiluminescence, and the relative expression of target proteins was normalized to β-Actin.