Qbox imaging system

Fresh frozen cerebral cortex was homogenized in RIPA buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 2 mM EGTA, 50 mM NaF, 0.5% SDS, 1% NP-40) containing a protease inhibitor cocktail (Roche Applied Science). Protein concentrations were determined by BCA (Thermo Scientific). Proteins were separated on an SDS-PAGE gel, and transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked in TBS (100 mM Tris-Cl, pH 7.5, 150 mM NaCl) containing 0.1% Tween-20, and 5% nonfat milk solution for 1 h, then incubated with one of the following primary antibodies: mouse-anti-PSD95 (1:1000; EMD Biosciences), mouse-anti-synaptophysin (1:500; Sigma), or mouse anti-βIII-Tubulin (1:2000; Sigma) overnight at 4 °C on a rotary shaker. Membranes were washed three times with 0.1% TBS prior to addition of an anti-mouse IgG horse radish peroxidase secondary antibody (1:2000; Cell Signaling Technology). Proteins were visualized by chemiluminescence (Millipore) on a QBOX imaging system (Syngene). Densitometry analysis was performed in ImageJ (NIH) using the gel analyzer plug-in.

Primary neuron, astrocyte or astrocyte derived EV pellets were lysed using RIPA buffer (50 mM Tris‐Cl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 2 mM EGTA, 50 mM NaF, 0.5% SDS, 1% NP‐40) with the protease inhibitor cocktail (cOmplete, Sigma‐Aldrich) and phosphatase inhibitor (phosphoSTOP, Sigma‐Aldrich). Protein concentrations were determined using the BCA protein assay reagent kit (Thermo Fisher Scientific). Equal amount of proteins was loaded into each lane, and proteins were separated by SDS‐PAGE, then transferred to nitrocellulose membranes using iBlot 2 Dry Blotting System (Thermo Fisher Scientific). After blocking for 1 h at room temperature with 5% nonfat milk (Bio‐rad), the membranes were incubated overnight at 4°C with primary antibodies in 5% nonfat milk. A full list of primary antibodies can be found in Table S3. Membranes were washed in TBST (TBS contain 1% Tween‐20) and exposed to the appropriate horseradish peroxidase‐conjugated secondary antibody (1:2000, Cell Signaling Technology) for 1 h. Immunoreactive proteins were visualized by chemiluminescence (Millipore) using a QBOX imaging system (Syngene), and quantified using ImageJ version 1.50i.