Ab227310

Samples (lung tissue or lung vascular
endothelial cells) were dissolved in lysis buffer for lysis, centrifuged
at 12,000g for 5 min to collect their supernatants,
and analyzed for their protein concentration using a BCA kit. High-resolution
separation of protein samples (20 or 40 μg protein/well) was
obtained on a 12% SDS-PAGE gel. These proteins were then transferred
to nitrocellulose or PVDF membranes (Milipore). These membranes were
blocked for 10 min at room temperature using a commercial blocking
solution (PS108, Epizyme Biotech, Shanghai, China), washed three times
with TBST, and incubated with VE-cadherin (1:1000, ab205336, Abcam),
ZO-1 (1:1000, ab216880, Abcam), ICAM-1 (1:1000, ab222736, Abcam),
β-catenin (1:20000, Affinity), ITGAM (1:1000, #17800, CST),
ITGB2 (1:1000, #72607, CST), TSG101 (1:1000, ab125011, Abcam), CD9
(1:1000, A1703, Abclonal), Calnexin (1:1000, ab227310, Abcam), and
Syntenin (1:1000, ab19903, Abcam), overnight at 4 °C (approximately
12 h). These membranes were then washed three times with TBST and
incubated with a fluorescently labeled secondary antibody (antirabbit
or antimouse lgG, Cell Signaling Technology) for 60 min at room temperature.
After three washes, the prints were visualized using an imaging system
(Bio-Rad).

Exos or PC3 cells were lysed with protease inhibitor-contained radio-immunoprecipitation assay buffer (Beyotime) and the supernatant was collected after a 20-min centrifugation at 12,000×g at 4°C, followed by determination using bicinchoninic acid kits (Sigma-Aldrich). Following separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Thermo Fisher Scientific), proteins were transferred onto polyvinylidene fluoride membranes. Membranes were next incubated overnight at 4°C with primary antibodies: anti-CD63 (1:1000, ab134045, Abcam, Cambridge, UK), anti-CD9 (1:1000, ab236630, Abcam), anti-CD81 (1:1000, ab219209, Abcam), anti-calnexin (1:500, ab227310, Abcam), anti-cyclin D (1:1000, ABE52, Merck Millipore, Billerica, MA, USA), anti-cyclin E (1:1000, PA5–16237, Invitrogen). Subsequently, membranes were rinsed and probed for 1 h with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (IgG, 1:2000, ab6721, Abcam) under room temperature, followed by detection with enhanced chemiluminescence. The gray analysis of bands was conducted with Image J software (NIH, Bethesda, MD, USA), with β-actin (1:200, ab115777) as an internal reference.