Non fat milk

Cell pellets were lysed in a Radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl pH 8.8, 150 mM NaCl, 1 mM Ethylenediaminetetraacetic acid, EDTA, 0.1% Sodium lauryl sulfate, SDS, 1% Triton X-100) containing protease inhibitors (Roche, Monza, Italy), incubated on ice for 30 min, and centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was collected and used for protein quantification by Bradford assay (Bio-Rad, Milan, Italy). For each sample, 30 μg of protein lysate was electrophoresed in SDS gel (12% acrylamide) and blotted onto a nitrocellulose membrane. The transferred membrane was blocked with 5% non-fat milk (Bio-Rad) in tris-buffered saline (TBST) buffer (100 mM Tris-HCl pH 8, 1.5 M NaCl, 0.1% Tween) for 1 h at room temperature (RT) and incubated with primary antibodies (anti-EGR1, Immunological Sciences AB-83620, 1:1000, and anti-GAPDH, Immunological Sciences MAB-91903, 1:5000) in TBST with 3% non-fat milk (Bio-Rad) overnight at 4 °C. After several washes with TBST, the membrane was incubated with the corresponding secondary antibodies (anti-rabbit, Bethtyl A120–108P, 1:5000, and anti-mouse, Bethyl A90–117P, 1:5000) in TBST with 3% non-fat milk (Bio-Rad). After several washes, immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection kit (EuroClone) according to the manufacturer’s instructions.

Tissue samples were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors, following standard procedures. After protein determination, using BioRad protein assay (BioRad, Milan, Italy), equal amounts of proteins (40 μg) were resolved on 8% SDS-PAGE gel and transferred to a nitrocellulose membrane (BioRad). Membranes were blocked with 3% nonfat milk (BioRad) in PBS Tween 0.05% (PBST) and incubated overnight at 4°C with the following antibodies: anti-EGFR (undiluted, Novocastra), anti-Beclin1 (1 : 270, Sigma-Aldrich), and anti-β-actin (1 : 500, catalogue number: 04-1116, MERCK Millipore Corporation, Billerica, MA, US) diluted with 3% nonfat milk in PBST. Membranes were washed three times in PBS Tween 0.1% and incubated with specific secondary antibodies diluted with 3% nonfat milk in PBST (goat anti-rabbit IgG (H + L)-HRP conjugate, diluted 1 : 10000, catalogue number: 172-1019, BioRad; goat anti-mouse IgG (H + L)-HRP conjugate, diluted 1 : 5000, catalogue number: 172-1011, BioRad) for 1 h at RT. The membranes were incubated with ECL reagents (BioRad) for 1 min and then were developed on Hyperfilm ECL (Amersham GE Healthcare, 28906835).
Images of the bands were digitized and the densitometry was performed using the open source image processing program ImageJ (http://imagej.nih.gov/ij/); β-actin bands were used for normalization.

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.