Immobilon

NSCs were incubated in NSC media without B27, N2, EGF, and FGF growth supplements for 2 hours prior to a brief PBS wash and centrifugation. Cell pellets were lysed in cold lysis buffer (20 mM Tris [pH 7.5], 10 mM EGTA, 40 mM β-glycerophosphate, 1% Nonidet P40, 2.5 mM MgCl₂, and 2 mM sodium orthovanadate) supplemented with aprotinin (1.4 μg/ ml), leupeptin (1 μg/ml), and phenylmethanesulfonylfluoride (1 mM) protease inhibitors for protein quantitation using the Pierce BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, www.thermofisher.com). The resulting cell lysates (20 μg) were separated by SDS-PAGE followed by transfer to polyvinylidene difluoride membranes (Immobilon, Thermo Fisher). Membranes were blocked with 5% milk in 1× TBS-Tween prior to overnight incubation at 4 °C with appropriate primary antibodies (Supporting Information Table S2). Detection was achieved using species-appropriate HRP-linked secondary antibodies (Cell Signaling, Danvers, MA, www.cellsignal.com) and enhanced chemiluminescence imaging (Bio-Rad Laboratories, Hercules, CA, www.bio-rad.com) using the ChemiDoc-It Imaging System (UVP, Upland, CA, www.uvp.com).

Exosomes were resuspended in radioimmunoprecipitation assay buffer (Thermo Scientific, Waltham, MA) containing Halt protease inhibitor cocktail (Thermo Scientific) and the protein concentration was determined by Bradford analysis. For western blotting, the lysate was run on a 12% SDS-PAGE (NuPAGE, Novex/Life Technologies) for 1.5 h at 150 V. After 1 h transfer at 100 V, the nitrocellulose membrane was blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween20 for 1 h, incubated with rabbit anti-HSP70 (Abcam, Cambridge, UK) or rabbit anti-TSG101 (Genetex, Irvine, CA) overnight and then with goat anti-rabbit IgG-HRP (System Biosciences, Mountain View, CA). LPS was detected after 1 h incubation with mouse anti-LPS (Abcam, ab35654) and goat antimouse IgG-HRP (Abcam, ab6789). Proteins were detected by Immobilon (Thermo Scientific) detection reagent and film exposure. For cytokine array, 100 μg of protein was incubated with the blots according to manufacturer’s specifications (R&D Systems, Minneapolis, MN).