Lamin b1

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.

For western blotting, 20–35 μg protein was separated on 6% or 10% polyacrylamide gels and transferred on polyvinylidene fluoride membranes using the TransBlot Turbo system (Bio-Rad). For antibody-based staining, the membrane was washed once with TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl and 0.1% Tween-20), blocked with 5% non-fat dry milk in TBS-T and stained with primary antibodies against ATF7IP (Bethyl, A300-169A), ZMYM2 (Bethyl, A301-711A-M), or LAMIN B1 (Santa Cruz Biotechnology, 374015) at 4 °C overnight. Next day, membranes were washed three times with TBS-T for 10 min before incubation with species-specific horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After additional three washes with TBS-T for 10 min each, signal was detected using the Amersham ECL Western blotting detection reagent (GE Healthcare Life Sciences; RPN2109) and exposure on Amersham Hyperfilm ECL (GE Healthcare Life Sciences; 28906836) in a darkroom.