Anti cd63

Plasma sEVs (10 μg) in non-reducing (CD63 only) or reducing sample buffer were separated on 4–20% polyacrylamide gels (Bio-Rad, #4561094) and transferred onto nitrocellulose membranes (Bio-Rad, #1620090). Briefly after blocking, the membrane was incubated with the following primary antibodies overnight at 4 °C: anti-CD63 (Invitrogen, #10628D, 1:250), anti-CD9 (Invitrogen, #10626D, 1:500), anti-TSG101 (Invitrogen, #PA5-31260; 1:500), anti-Grp94 (CST, #2104; 1:1000 in 5% BSA in PBS), anti-ApoA1 (CST, #3350; 1:1000). After washing, HRP-conjugated secondary antibodies (IgG Rabbit anti-Mouse, Invitrogen, #31450, 1:10,000 or IgG Goat anti-Rabbit, Invitrogen, #31460, 1:10,000) were added and incubated for 1 h at RT. The chemiluminescence signal was elicited by SuperSignal™ West Dura™ Chemiluminescence Substrate (Thermo Scientific, #34076) according to the manufacturer’s instructions.

Proteins were prepared with a detergent buffer (R0278, Sigma-Aldrich), and the protein concentration was determined using the Pierce™ BCA Protein Assay Kit (20164, Thermo Fisher Scientific)y. Equal amounts (60 μg) of protein samples were separated by a 12% gel using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 1610174, Bio-Rad Laboratories, Shanghai, China) and transferred onto polyvinylidene fluoride (PVDF; IPVH00010, Millipore, Bedford, MA, USA) membranes. After block by skim milk (37587, Thermo Fisher Scientific) for 1 h, the samples were then incubated with respective primary antibodies including anti-RNF157 (WH0114804M1), anti-PLRG1 (SAB2500805), anti-SMU1 (SAB1407636), anti-CHD1 (ZRB1692) and anti-PSMD8 (SAB1406325) provided by Sigma-Aldrich, and anti-β-actin (PA1-46296), anti-CD63 (MA1-19281), anti-TSG101 (1062BD), anti-calnexin (PA5-34665), anti-HDAC1 (49-1025), anti-RAN (48-2300), anti-EMD (701503) and anti-FXR2 (MA1-5773) provided by Invitrogen overnight at 4°C, followed by cultivation with secondary antibodies marked by horseradish peroxidase (HRP) (32260, Invitrogen) at room temperature for 2 h. Eventually, enhanced chemiluminescence (ECL) detection system (32209, Thermo Fisher Scientific) was applied for gray-scale value analysis.

Eluted EVs were lysed in 10X cell lysis buffer (Cell Signaling Technology, Germany) with protease inhibitor cocktail B (Santa Cruz, United States of America) and were mixed with Roti-Load 2 (Carl Roth, Germany). For reducing conditions DTT was added. 24 μl of the sample was loaded on a 10% or 12% SDS-polyacrylamide electrophoresis gel and transferred to a nitrocellulose membrane. Subsequently, it was blocked in 5% non-fat dry milk and incubated with anti-CD63 (Invitrogen, Germany) and anti-CD81 (Invitrogen, Germany) under non-reducing conditions, anti-Flotillin-1 (BD Biosciences, United States of America) and anti-Calnexin (Cell Signaling Technology, United States of America) under reducing conditions. Afterwards, the membrane was thoroughly washed and incubated with the secondary antibody coupled with a horseradish peroxidase. The chemiluminescence was detected using the ECL Plus Western Blotting Detection Reagent (Amersham, United States of America) on the Luminescent Image Analyzer LAS-3000 (FUJIFILM, Japan).

EVs were placed on Formvar-carbon coated nickel grids for 1 h, washed three times with PBS, and fixed with 2% paraformaldehyde for 10 min. After three washes, grids were then incubated for 2 h with the following antibodies: anti-CD63 (10628D, Invitrogen, Thermo Fisher Scientific) and anti-CD81 (10630D, Invitrogen). EVs were then washed five times and incubated with a 10 nm-gold labeled secondary antibody. They were washed five more times and post-fixed with 2.5% glutaraldehyde for 10 min. Samples were contrasted using 2.5% uranyl acetate for 10 min, followed by four washes and an incubation of 10 min in lead citrate. Grids were finally washed four times in deionized water and examined with a JEOL JEM-1400 transmission electron microscope at 80 kV.

Protein concentrations of sEV samples were measured using a BCA protein assay (Pierce Biotechnology, Waltham, MA, USA). Proteins were separated by 12% SDS-PAGE in reducing or non-reducing conditions and 10 ug protein aliquots/lane were transferred onto a PVDF 0.2 µm membrane (Millipore, Burlington, MA, USA) followed by blocking with 5% non-fat milk. Incubation with primary antibodies anti-CD63 (1:400, Invitrogen, Waltham, MA, USA, 10628D), anti-CD9 (1:1000, Invitrogen, 10626D), and anti-Grp94 (1:1000, Thermo Fisher, 36-2600) was performed overnight at 4 °C, followed by incubation with secondary HRP-conjugated antibody (1:1000 in 5% non-fat milk, anti-rabbit, anti-mouse, Cell Signalling Technology, Danvers, MA, USA) for 1 h at RT. Visualization was performed by chemiluminescence ChemiDoc.

The exosomes were lysed in PRO-PREP (Intron Biotechnology, Seoul, South Korea) according to the manufacturer’s protocol. The protein was electrophoresed on 12% SDS-PAGE gel and was transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skim milk in T-TBS (10 mM Tris, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature and was subsequently incubated with anti-CD9, anti-CD63, or anti-CD81 (Invitrogen, Carlsbad, CA, USA) (see Table 1 for details on antibodies) overnight at 4 °C. After vigorous washing in T-TBS, the blots were incubated with horseradish peroxidase (HRP)-tagged anti-rabbit or anti-mouse secondary antibodies from Santa Cruz Biotechnology (Dallas, TX, USA) for 1 h. The labeled proteins were visualized using the ChemiDoc™ XRS imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using an enhanced chemiluminescence kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA). All chemical reagents were purchased from Bio-Rad Laboratories (Hercules, CA, USA).

SEV samples (5–10 μg) in non-reducing (CD63 and CD81) or reducing sample buffer were separated on 4–20% polyacrylamide gels (Bio-Rad) and transferred onto nitrocellulose membranes (Bio-Rad). Briefly, after blocking, the membrane was incubated with the following primary antibodies overnight at 4 °C according to manufacturer’s instructions: anti-CD63 (#10628D, 1:250), anti-CD9 (#10626D, 1:500), anti-CD81 (#10630D, 1:500), anti-TSG101 (#PA5-31,260, 1:500) from Invitrogen; anti-PD-L1 (#13,684, 1:1000), anti-amylase (#3796,1:1000), anti-TGF-β (#3711, 1:1000) from Cell Signaling Technology; anti-CTLA-4 (#TA810204, 1:500) from OriGene; anti-TRAIL (#ab2056, 1:500) from Abcam. After washing, HRP-conjugated secondary antibodies (IgG Rabbit anti-Mouse, 1:10,000 or IgG Goat anti-Rabbit, 1:10,000; Thermo Scientific) were incubated for 1 h at RT. The chemiluminescence signal was elicited by SuperSignal™ West Dura™ Chemiluminescence Substrate. Images were acquired with the iBright Imager. To compare the total protein content of SEVs, 35 µL of the samples were loaded and gels were stained using Coomassie blue (Thermo Scientific).