Odyssey gel imaging system

Total exosomic and cellular protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Beijing, China). The protein concentrations of cell and exosome lysates were determined using BCA protein assay kit (Beyotime Institute of Biotechnology, Beijing, China). For Western blot analyses, the cellular and exosomic proteins (10 µg/well) were separated by electrophoresis in 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in TBS-T (10 mM TrisCl, pH 8.0, 150 mM NaCl, 0.5% Tween 20) at 4°C overnight, rinsed 3 times (10 min/time) with TBS-T, followed by 3 h incubation at room temperature with the specific primary antibodies, followed by 1 h incubation with the second antibody. ECL developer (Thermo Scientific) was added and exposure imaging was performed by Odyssey gel imaging system. The specific antibodies to E-cadherin (ab1416), N-cadherin (ab76011), Vimentin (ab92547), Snail (ab216347), CD81 (ab109201), HSP70 (ab181606), TSG101 (ab125011), Calnexin (ab133615), were purchased from Abcam (Cambridge, UK). β-actin antibody (#4970) was acquired from Cell Signaling Technology (Danvers, MA, USA).