Prohibitin

Nanoparticle tracking analysis was performed to determine the size and particle concentration of EVs using an NS300 (Malvern Panalytical, Malvern, UK). Briefly, the samples were appropriately diluted (1:100–1:10,000) in particle-free phosphate buffer saline to achieve suitable concentrations. For immunoblotting, cells lysed in RIPA buffer or isolated exosomes were electrophorized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. Thereafter, the membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and incubated with primary antibodies against target protein (srIκB, CRY2), exosome positive marker, and exosome negative marker overnight at 4 °C. The following primary antibodies were used in immunoblotting studies: srIκB, CRY2 (customized antibody from Abclon, Seoul, Republic of Korea), CD9, CD81 (SBI, Tokyo, Japan), TSG101, alix, GM130, calnexin (Abcam, Cambridge, UK), lamin B1, GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), and prohibitin (NOVUSBIO, Centennial, CO, USA). Following incubation with specific secondary antibodies, the blots were developed using Clarity and Clarity Max ECL western blotting substrates and visualized using a ChemiDoc imager.

The morphology and lipid bilayer of extracellular vesicles (EVs) were absorbed on carbon-coated copper, stained with 2% uranyl acetate, and confirmed by transmission electron microscopy. Nanoparticle tracking analysis was used to measure EVs’ particle number and size distribution using NS300, and samples were diluted (1:100–1:10,000) in particle-free PBS to an acceptable concentration. Immunoblotting was performed by lysing cells in RIPA buffer or exosomes, followed by SDS/PAGE gel electrophoresis and transfer onto nitrocellulose membrane. Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and probed with primary antibodies against srIκB, CRY2 (customized antibody, AbClon, Seoul, Korea), CD9, CD81 (SBI, Tokyo, Japan), TSG101, Alix, GM130, calnexin (Abcam, Cambridge, UK), lamin B1, GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), and prohibitin (Novusbio, Centennial, CO, USA) at 4 °C overnight. After incubation with specific secondary antibodies, blots were developed using Clarity and Clarity Max ECL Western blotting substrates and imaged with the ChemiDoc imager.