Anti cd9

Lysates of cells or exosomes were diluted at a ratio of 1:5 with protein loading buffer (5×; Beyotime Biotechnology, Jiangsu, China) and heated at 95 °C for 5 min. Protein extracts were separated by SDS-PAGE (6% gel for DMBT1 and 12% gel for other proteins) and transferred to polyvinylidene fluoride membranes (Immobilon P, Millipore, USA). The membranes were blocked with 5% milk in TBST (Tris-buffered saline, 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 60 min at room temperature, incubated with primary antibodies at 4 °C overnight and then incubated with the horseradish peroxidase-conjugated secondary antibodies at 37 °C for 1 h. Primary antibodies and dilutions were used as follows: anti-CD9 (1:500; Santa Cruz), anti-CD63 (1:300; Santa Cruz), anti-CD81 (1:500; Santa Cruz), TSG101 (1:1000; ProteinTech, Chicago, USA), anti-DMBT1 (1:5000; Abcam, Cambridge, Britain), anti-VEGF-A (1:500; R&D system, Minneapolis, USA), Akt (1:500; Cell Signaling Technology, Danvers, USA), anti-phosphorylate Akt (p-Akt; 1:500; Cell Signaling Technology) and anti-GAPDH (1:5000; Cell Signaling Technology). All the secondary antibodies (1:5000) were obtained from Cell Signaling Technology. The immunoreactive bands were visualized using enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, USA) and imaged by the ChemiDoc XRS Plus luminescent image analyser (Bio-Rad).

The concentration of proteins in the analyzed samples was assessed using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, USA; 23 225) according to the manufacturer’s instructions. Twenty microliters of SEC fractions (corresponding to ~5–6 μg of proteins) were mixed with loading buffer to a final concentration of 2% (v/v) SDS, 0.1% (v/v) bromophenol blue, 10% (v/v) glycerol, and optionally 100 mM DTT, denatured for 5 min at 95°C and separated by 12% SDS-polyacrylamide gel electrophoresis followed by wet transfer onto nitrocellulose membranes (Thermo Fisher Scientific, Waltham, USA; 88 018). Membranes were blocked for 1 h in 5% non-fatty milk and 0.1% Tween in PBS, and then primary antibody (anti-CD63: Invitrogen, 10 628D, 1:1500; anti-CD9: Santa Cruz Biotechnology, sc-13 118, 1:800; anti-CD81: Invitrogen, 10 630D, 1:500) was added for overnight incubation at 4°C. After triplicate washes, secondary antibody conjugated with HRP was added for 1 h at room temperature. Chemiluminescence detection of bands was performed with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, USA; 34 095) diluted 1:10 with washing buffer. CD63 and CD81 were detected under non-reducing conditions.