Tsg101

Details for sEV western blot were previously described (55 (link)). To assess sEV purity, the presence of the following markers was assessed: Alix (i.e., PDCD6IP, #2171, RRID:AB_2299455, Cell Signaling Technology), TSG101 (#GTX70255, RRID:AB_373239, GeneTex), CD63 (#556019, RRID:AB_396297, BD Biosciences), and CD81 (#sc-7637, RRID:AB_627190, Santa Cruz Biotechnology). Furthermore, the absence of two common contaminating proteins, Calnexin (#2433, RRID:AB_2243887, Cell Signaling Technology) and PHB (#sc-377037, RRID:AB_2714190, Santa Cruz Biotechnology), was also evaluated.

Protein concentrations of EV samples were determined using the micro-bicinchoninic acid assay (Thermo Fisher, Waltham, USA). Western blots were performed with 5 μg of the concentrated EV fractions, which were treated with sample buffer (dithiothreitol, 0.1% SDS, 0.1 M Tris HCl; pH 7.0) and boiled for 5 min at 95 °C before separation on 12% SDS-polyacrylamide gel electrophoresis gels. The samples were transferred to polyvinylidene fluoride membranes (Merck Group, Darmstadt, Germany). The membranes were blocked by 5% milk solution (skim milk powder (Thermo Fisher, Waltham, USA) dissolved in Tris-buffered saline solution with 1% Tween-20) for 1 h at RT and stained with antibodies recognizing “exosomal marker” proteins including CD63 (1:500, Biorbyt, Cambridge, UK), TSG101 (1:500, GeneTex, Irvine, USA), Alix (1:500, BD Transduction Laboratories, San Jose, USA), and negative control calnexin (1:500, Abcam, Cambridge, UK) followed by a 1-h incubation with a matched horseradish peroxidase-labeled secondary antibody. Immunoreactivity was detected using chemiluminescence detection kit reagents and a Chemidoc Station (Biorad, Hercules, USA). ImageJ was used for densitometric analysis of each blot. The Western blotting procedures were repeated three times per sample. Please also refer to Supplementary Figure S2 for uncropped Western blots.

Brain homogenates (10 μg protein), fibroblast lysates (10 μg protein), and EVs proteins (15 μl of the lysate corresponding to 25% of the EVs lysate total volume), were separated by 4–20% tris-glycine gel electrophoresis (Criterion precast gel, Bio-Rad, Hercules, CA) and transferred onto PVDF membranes (Immobilon, EMD Millipore, Billerica, MA). Membranes were incubated with antibodies to CD63 (1:250, Cat# sc-15363, Santa Cruz Biotechnology, Dallas, TX), Alix (1:1000, Cat# ABC40, EMD Millipore), TSG101 (1:1000, Cat# 4A10, GeneTex, Irvine, CA), Flotillin-1 (1:1000, Cat# 610821, BD Biosciences, San Jose, CA), Flotillin-2 (1:1000, Cat# 610384, BD Biosciences), rab35 (1:1000, Cat# 9690, Cell Signaling Technology, Danvers, MA), and β-actin (1:2500, Cat# ab8226, Abcam, Cambridge, MA). The secondary antibodies used were HRP conjugated anti-rabbit, and anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). Protein bands were quantified using ImageJ software (NIH, Bethesda, MD). All data are shown as the trisomic to diploid ratio for Ts2 and 2N mice, or for DS patients and 2N control subjects.