Anti cd9

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.

sEV validation involved western blotting with Invitrogen's anti-CD63 (10628D), anti-TSG101 (MA-1-23296, Invitrogen), anti-CD9 (Invitrogen, PA5-86534), anti-Flotillin-1 (Invitrogen, PA5-17127), calnexin (E-AB-14819; Elabsciences), and anti-GAD65 (a marker for islets origin sEVs, PA5-22260, Invitrogen) antibodies. As the internal control, β-actin was used. The ImageJ program was used to quantify band intensities (NIH, USA). sEV cargo proteins, BIRC2/cIAP1 (DF6167, Affinity Bioscience), and Beclin-1 (E-AB-53242, Elabsciences) were assessed. sEVs were lysed through ultrasonication, and The Bicinchoninic Acid test (22802, ThermoFisher Scientific) was used to quantify total protein with BSA as the protein standard. Isolated sEV samples (10 μg) from control and patients were transferred to BioRad nitrocellulose membranes after being separated by 10% SDS PAGE. Further, Membranes were blocked in 3% BSA in TBS-T before primary antibodies (CD63, TSG10, anti-CD9, Flotillin-1, calnexin, GAD65, BIRC2/cIAP1, and Beclin-1) were added for overnight at 4 °C, further HRP-tagged secondary antibody (at room temperature for 2 h in the dark) was used. nitrocellulose membranes were stained using the G-biosciences' Femto LUCENT™ PLUS-HRP kit (786-003) for HRP-based electroluminescence and imaged using a GEL-Doc apparatus (Azure Biosystems).

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).

Western blot was performed as described previously [57 (link)]. Briefly, cell lysates or isolated EV samples (containing 10 μg protein) were resolved via 12.5% SDS-PAGE, transferred to PVDF membrane, and probed using anti-CD9 (catalog number: 10626D, Invitrogen) and anti-TSG 101 (catalog number: sc-7964, Santa Cruz Biotechnology). Subsequently, blots were incubated with horseradish peroxidase (HRP) conjugated secondary antibody (1:3000) for 1 h at room temperature. Washing steps after antibody incubations were conducted on an orbital shaker four times at 10 min intervals with TTBS. Blots were developed with chemiluminescent reagent (Pierce, Waltham, MA, USA). Original blots see Supplementary File S1.

MSC-EVs and cells were homogenized in RIPA buffer supplemented with a cocktail of protease and phosphatase inhibitors and phenylmethylsulphonyl fluoride (PMSF, Millipore Sigma, St. Louis, MO, USA). Protein concentration was measured by nicinchoninic acid (BCA) protein assay following the manufacturer’s instructions (Pierce, Thermo Fisher Scientific, Waltham, MA, USA). Five µg of protein extracts from EV or BM-MSCs were loaded onto 4–20% Criterion TGX Stain-Free Precast gels and transferred onto nitrocellulose membranes (BioRad, Hercules, CA, USA). The membranes were then incubated with the following primary antibodies: anti-CD9 (1:1000, Invitrogen, Carlsbad, CA, USA), anti-Alix (1:200; Santa Cruz, Santa Cruz, CA, USA), anti-RS29 (1:1000, Abcam, Cambridge, UK), anti TSG-101 (1:200; Santa Cruz, Santa Cruz CA, USA), anti-calnexin (1:1000, Abcam, Cambridge, UK), and the HRP-conjugated secondary antibodies: goat anti-rabbit (1:5000, Pierce, Thermo Fisher Scientific, Waltham, MA, USA), goat anti-mouse (1:5000, Pierce, Thermo Fisher Scientific, Waltham, MA, USA). Membranes were developed with SuperSignalWest Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Meridian Rd., Rockford, IL, USA), and signal captured on a ChemiDoc™ MP Imaging System (BioRad, Hercules, CA, USA).