Antiflotillin 1

Exosomal protein makers were determined using western blot. Fifteen micrograms of EVs protein were resolved with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE, Sigma‐Aldrich) and blotted to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Bedford, MA, USA). The blotted membrane was blocked with 5% skim milk (Sigma‐Aldrich) for 1 h at room temperature followed by staining with anti‐CD81 (1:250, cat. 10630D, Invitrogen, La Jolla, CA, USA) and antiflotillin‐1 (1:2000, cat. F1180, Sigma‐Aldrich) at 4 °C overnight. The membrane was washed with buffer several times to remove primary antibodies before staining with horseradish peroxidase (HRP)‐conjugated secondary antibody for 1 h at room temperature. The chemiluminescent signal was developed using ECL Prime western blotting detection reagent (GE Healthcare, Chicago, IL, USA) and visualized by chemiluminescent detection instrument (Bio‐Rad, Hercules, CA, USA).

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).