Dithiothreitol (dtt)

Western blot analysis was performed to determine extract-induced changes in the level of key proteins and their modifications. Cells were incubated with the extracts for 48 h, collected by centrifugation, washed with ice-cold PBS and lysed with lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Total protein concentration was determined using the BCA protein Assay kit (Thermo Scientific, Rockford, IL, USA). The protein extracts were combined with the loading buffer containing dithiothreitol (Cell Signaling Technology), boiled for 5 min, and aliquots of equal amount of proteins were separated on polyacrylamide-SDS gels by electrophoresis. The proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The relevant antibodies were added and detected using the chemiluminescent substrate Immobilon (EMD Millipore). The antibodies used, their dilutions and sources are presented in S1 Table.

Cells were lysed using the IP Lysis Buffer Protocol (Pierce, Waltham, MA) as described by the manufacturer. Biotinylated peptides (EV-b and NT-b, 40 μg) were incubated with 100 μL of streptavidin agarose resin at 4°C for 1 hour on a rotator to generate the peptide-coupled streptavidin agarose resin. The protein samples were pre-cleared by incubation of 50 μL of NT-streptavidin agarose resins (4°C on rotator for 1 hour). After centrifugation, the pre-cleared samples were incubated with 150 μL of EV-coupled streptavidin agarose resin at 4°C overnight on rotator. After PBS wash, the resin was pelleted and sent for mass spectrometry analysis (see details in Supplementary Methods) or eluted with 4x Laemmli Sample Buffer (Bio-Rad, Hercules, CA) containing 1X DTT reducing agent (dithiothreitol, Cell Signaling Technology, Danvers, MA) and boiled for SDS-PAGE. Pulled-down proteins were then separated with a Novex™ WedgeWell™ (ThermoFisher Scientific, Waltham, MA) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA). Membrane-transferred proteins were immunoblotted with an antibody recognizing N-cadherin (1/10000, NBP2-01498, Novus Biologicals, Littleton, CO) (Supplemental Figure 2).

vWF and D-Dimer in the plasma were measured by Western blot at age 5 months. Equal volumes of the plasma were loaded on the gel. Before loading, 3× Laemmli Sample buffer supplemented with dithiothreitol (no. 7722, Cell Signaling) was added to the samples and the mixtures were boiled for 5 minutes. Immunoblots used primary antibodies against vWF (no. A0082, Dako) and D-Dimer (no. bs-3514R, Bioss) and secondary antibodies labeled with Alexa Fluor 680 nm Dye (Invitrogen). The immunoblots were scanned and integral fluorescence from each band was measured using Odyssey Infrared Imaging System (Li-COR Biosciences). See complete unedited blots in the supplemental material. For final quantification, we corrected the measurements for plasma dilution with sodium citrate by multiplying by the coefficient of dilution calculated as described in the Mouse blood collection and plasma isolation section.