Exoab cd9a 1

sEV samples were lysed in ice-cold RIPA buffer (Beyotime, China) on ice for 15 min and centrifuged at 13,000 g for 10 min. The protein concentration in the supernatant was determined via the BCA assay (Pierce, NCI225CH). Thirty micrograms of total protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto a PVDF membrane (Millipore, USA). The membrane was blocked with 5% nonfat milk PBST for 1 h at RT and then incubated with primary antibodies against CD63 (rabbit polyclonal, System Biosciences, EXOAB-CD63A-1), CD81 (rabbit recombinant monoclonal, Abcam, ab109201), CD9 (rabbit polyclonal, System Biosciences, EXOAB-CD9A-1), calnexin (rabbit polyclonal, Proteintech, 10427-2-Ap) and albumin (rabbit polyclonal, Proteintech, 16475-1-AP) overnight. After incubation with a goat anti-rabbit HRP secondary antibody (Jackson Immunoresearch, West Grove, PA, 111-035-003) for 1 h at RT, protein bands were visualized using an enhanced chemiluminescent (ECL) substrate (Tanon, Shanghai, China, 180–501).

Dulbecco’s modified Eagle medium (DMEM; #08458-45) and RPMI-1640 were obtained from Nacalai Tesque (Kyoto, Japan). Fetal bovine serum (FBS; #172012) and hepatocyte growth factor (HGF; #H1404) were procured from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against c-Met (C-28; #161), E-cadherin (#8426), and cytokeratin-18 (#6259) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against CD9 (EXOAB-CD9A-1), CD63 (EXOAB-CD63A-1), CD81 (EXOAB-CD81A-1), and α-SMA (#14395-1-AP) were obtained from Proteintech (Rosemont, IL, USA). Antibodies against β-actin (#4967), phospho-AKT (Ser473; #9271), extracellular signal-regulated kinase (ERK) 1/2 (3A7; #9107), phospho-ERK 1/2 (p-ERK 1/2; Thr202/Tyr204, E10; #9106), and AKT (#9272) were purchased from Cell Signaling Technology (Danvers, MA, USA). Lipofectamine 3000 (#L3000008), TRIzol (#15596-018), and anti-phospho-c-Met antibody (pypypy1230/1234/1235; #44888G) were obtained from Life Technologies (Carlsbad, CA, USA). The antibody against vimentin (#M0725) was procured from DAKO (Glostrup, Denmark). Streptavidin 10 nm gold (#AC-10-04-05) was purchased from Cosmo Bio (Tokyo, Japan).

For western blot analysis, exosomes were lysed in RIPA buffer (#9806, Cell Signaling Technology) with Protease Inhibitor Mixture (Roche Diagnostics, Germany) and quantified using a BCA protein assay reagent kit (Pierce, USA). The protein samples were denatured at 95°C for 10 min in 5 × Laemmli buffer. Then, 20 μg of protein was separated by 10% or 12% SDS-PAGE and transferred onto Hybond-C Extra membranes (Amersham Biosciences, Piscataway, NJ). After blocking with 5% skim milk, the membranes were incubated with primary antibodies, including anti-CD9 (System Biosciences, EXOAB-CD9A-1, 1:10,000), anti-CD81 (Proteintech, 18250-1-AP, 1:1000), anti-calnexin (Proteintech, 10427-2-AP, 1:5000), anti-PSMA3 (Santa Cruz Biotechnology, sc-166205, 1:1000), and anti-PSMA6 (Santa Cruz Biotechnology, sc-271187, 1:1000) overnight. Then, the membranes were incubated with a secondary antibody (Cell Signaling Technology, 1:5000) for 1 h at room temperature. Each step was followed by washing in 1 × TBS-T for 10 min 3 times each. The immunoreactive blots were visualized using a chemiluminescence kit (Millipore, Billerica, MA) and imaged with a Tanon 5200 Multi-imaging system (Tanon, Shanghai, China). Densitometric analysis was performed using the western blot analysis images using Gel-Pro Analyzer software (Media Cybernetics, United States).