Anti cd81

A total of 40 µg of total protein of exosome lysate were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with Tris-Buffered Saline solution containing 0.05% Tween (TBS-T) and 5% non-fat dry milk and incubated overnight with the primary antibodies anti-CD9 (# MA1-80307, 1:5,000; Invitrogen, Thermo Fisher, MA, USA), anti-CD63 (# sc-5275 1:1,000; Santa Cruz Biotechnology, CA, USA) and anti-CD81 (# MA5-13548; Invitrogen, Thermo Fisher, MA, USA) at 4 °C. Then, the membranes were washed three times with TBS-T and incubated with HRP conjugated secondary antibodies (1:30,000 donkey anti-rabbit IgG-HRP sc-2313; 1:50,000 donkey anti-mouse IgG-HRP sc-2314; InvitrogenTM; Thermo Fisher, MA, USA) for 90 min. The membranes were washed again and incubated with an enhanced chemiluminescence substrate (SuperSignal™ West Femto Maximum Sensitivity Substrate; ThermoFisher Scientific, MA, USA). The immunoreactivity signal was revealed by chemiluminescence (ChemiDoc™; Bio-Rad Laboratories, Inc., CA, USA).

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.

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).

Cells were washed with PBS and scraped into a RIPA lysis buffer (50 mM Tris (pH = 7.5), 150 mM NaCl, 10 mM CaCl₂, 0.5% NP-40, 0.25% sodium desoxycholate, 0.1% SDS, protease inhibitor cocktail cOmplete Mini EDTA free (Roche), and 0.2% octylglucoside). Cell debris was removed by centrifugation at 10,000× g for 15 min. EVs were lysed using a 1% Triton X-100/0.1% SDS buffer. Protein concentrations were measured using the BCA Protein Assay kit (Pierce), following the manufacturer’s instructions. A total of 20 µg of protein-lysates were resolved using SDS-PAGE and transferred to polyvinlidene fluoride membranes (Millipore). Blots were blocked for 1 h with 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBST). Then, blots were probed overnight at 4 °C with the following primary antibodies: anti-CD63 (556019, BD Biosciences, Erembodegem, Belgium), anti-CD9 (sc-20048, Santa Cruz Biotechnology), anti-CD81 (10630D, Invitrogen), anti-CD31 (M0823, Dako), and anti-α-SMA (M0851, Dako). After three washes with TBST, blots were incubated for 1 h with a peroxidase-conjugated secondary antibody (7076, Cell Signaling, Bioké, Leiden, The Netherlands). The detection was performed using a chemilunescent system (ECL; Pierce, Thermo Fisher Scientific, Merelbeke, Belgium).

Exosome and cellular proteins were obtained by fractionation of cell lysates with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The protein bands were transferred onto a polyvinylidene fluoride membrane (PVDF) and incubated overnight at 4 °C with the following antibodies: rabbit monoclonal anti-CD9 (Abcam, Cambridge, UK), anti-CD63 (Abcam), anti-calnexin (Abcam), anti-CD81 (Invitrogen, MA, USA), anti-TSG101 (Invitrogen), and anti-GM130 (Cell Signaling, MA, USA). Before probing, nonspecific binding was blocked by incubation with 5% skim milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.5% Tween-20) for 60 min at room temperature. Membranes were washed four times for 10 min each and incubated with horseradish peroxidase-linked goat anti-rabbit secondary antibody (1:3000; Abcam) at room temperature for 1 h. Blots were washed four times with TBST and developed with the enhanced chemiluminescence system (Amersham Biosciences, Waltham, MA, USA) according to the manufacturer’s protocols and were visualized using the Chemidoc imaging system (BioRad, Hercules, CA, USA).