Xp00105box

The concentrated media/ACSF with extracellular vesicle (EV) depletion were prepared in 4x Laemmli buffer and then boiled for 5 min, followed by SDS/PAGE (10% Tris-Glycine gel; XP00105BOX, Invitrogen), then transferred to a nitrocellulose membrane (10600001, Amersham). After blocking in TBST buffer (20 mM Tris-HCI, 150 mM sodium chloride, 0.1% Tween-20) containing 5% (wt/vol) nonfat dry milk for 1 h at room temperature, the membrane was incubated with primary antibodies overnight at 4°C, then with secondary antibodies for 1 h at room temperature. The following antibodies were used: Tau5 (ab80579, Abcam), AT8: anti-phospho-Tau pSer202/Thr205 (MN1020, ThermoFisher Scientific), PHF-1: anti-phospho-Tau pSer396/Ser404 Tau (from Dr. Peter Davies), β-actin (4967S, cell signaling), anti-Tubulin (ab4074, Abcam). IRDye 800CW goat anti-mouse IgG secondary antibody (P/N: 926–32210, LI-COR), IRDye 680CW goat anti-rabbit IgG secondary antibody (P/N: 926–68071, LI-COR). Membranes were visualized by Odyssey Infrared Imager (model 9120, LI-COR Biosciences), and relative optical densities of bands determined by Fiji/ImageJ software.

The concentrated media/ACSF with extracellular vesicle (EV) depletion were prepared in 4x Laemmli buffer and then boiled for 5 min. Samples were either dotted directly onto nitrocellulose membranes for dot blotting or separated by SDS/PAGE (10% Tris-Glycine gel; XP00105BOX, Invitrogen) and then transferred to a nitrocellulose membrane (10600001, Amersham). After blocking in TBST buffer (20 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween-20) containing 5% (wt/vol) nonfat dry milk for 1 h at room temperature, the membrane was incubated with primary antibodies overnight at 4 °C, then with secondary antibodies for 1 h at room temperature. The following antibodies were used: Tau5 (ab80579, Abcam), AT8: anti-phospho-Tau pSer202/Thr205 (MN1020, ThermoFisher), PHF-1: anti-phospho-Tau pSer396/Ser404 Tau (from Dr. Peter Davies), p-GR (4161 S, Cell Signaling), GR (12041 S, Cell Signaling), β-actin (4967 S, cell signaling), anti-Tubulin (ab4074, Abcam). IRDye 800CW goat anti-mouse IgG secondary antibody (P/N: 926-32210, LI-COR), IRDye 680CW goat anti-rabbit IgG secondary antibody (P/N: 926-68071, LI-COR). Membranes were visualized by Odyssey Infrared Imager (model 9120, LI-COR Biosciences), and relative optical densities of bands determined by Fiji/ImageJ software. Full immunoblots used in the figures of this manuscript are shown in Fig. S4.

For western blotting, a total of 10 μg of protein was run on Novex 10% Tris-Glycine mini gels (ThermoFisher XP00105BOX). Protein was transferred to a PVDF membrane (Bio-Rad 1620177) using the Pierce G2 Fast Blotter. Blots were probed according to the supplier’s recommended protocol for the following antibodies: anti-actin (Sigma A2066), anti-FLAG (Sigma F1804), and anti-StrepII (MBL International catalog #M211–3). Secondary antibodies (anti-mouse IgG-peroxidase and anti-rabbit IgG-peroxidase; Sigma A0168 and A0545, respectively) were used at 1:10,000 dilution. Signal was detected using SuperSignal West Pico Chemiluminescent Substrate kit (ThermoFisher 34,078), exposed to Carestream Biomax XAR Film (Sigma 1,651,454), and developed using a Konica SRX-101A tabletop processor.