Modified lowry assay

EVs were lysed in Winman’s buffer (1% NP-40, 0.1 M Tris–HCl pH 8.0, 0.15 M NaCl, and 5 mM EDTA [Ethylenediaminetetraacetic acid]) with EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland), and quantified using a modified Lowry assay (Bio-Rad, Hercules, CA, USA). After quantification, 5 μg of proteins was separated on a 12% Bis–Tris SDS-PAGE gel and transferred into a nitrocellulose membrane (GE Healthcare, Cleveland, OH, USA). Membranes were then incubated with the anti-HSP70, anti-annexin-XI, anti-CD63, anti-syntenin, and anti-cytochrome c primary antibody. Signals were detected using the ECL Western blot Detection.

To assess mTORC1 activation in the whole hippocampus, mice were sacrificed at 4, 24, 48, and 72 h and 1 week after CCI or after sham surgery. The ipsilateral hippocampi were collected, homogenized with ice-cold Triton lysis buffer (1% Triton X-100, 20 mm Tris-HCL, 150 mm NaCl, 5 mm EGTA, 10 mm EDTA, and protease inhibitor cocktail [Roche, Basel, Switzerland]), and centrifuged for 30 min at 14,000 rpm, 4°C. Protein concentration was determined by a modified Lowry assay (Bio-Rad, Hercules, CA). The same amount of protein in each sample was loaded and run on SDS/PAGE. After electrotransfer to nitrocellulose membranes at 30 V overnight at 4°C, the membrane was incubated in PBS with 5% nonfat milk at room temperature for 1 h and probed with antibodies against S6 (1:1000, rabbit, Cell Signaling Technology), β-actin (1:000, mouse, Abcam), and pS6 (1:200, rabbit, Cell Signaling Technology) overnight at 4°C. Secondary antibodies were used at a dilution of 1:5000. The membrane was washed three times and rinsed in TBST. Finally, proteins were detected with ECL substrates (Bio-Rad), and images were acquired using normal image scanning methods for colorimetric detection.

HCT116 tumours harvested from mice were frozen in liquid nitrogen and then stored at −80°C. About 300 mg of sample was collected from frozen tissues and subsequently transferred to cork‐sealed homogenizing tubes containing a homogenizing ball (Mp Biomedicals LLC.) and 500 μl of RIPA buffer containing phosphatase and protease inhibitor cocktail (1 ml of RIPA buffer + 10 μl cocktail of protease inhibitors + 10 μl cocktail of phosphatase 1 inhibitors + 10 μl of phosphatase 2 inhibitors). All reagents were purchased from Sigma‐Aldrich Chemie GmbH. Homogenization of tissues was done using the Fast Prep®‐24 MP Bio homogenizer (Mp Biomedicals) with the following settings: CP 24 × 2, 6 m/s, t = 40 s, and the homogenization cycle was repeated twice. The resulting suspension was incubated on ice for 20 min, then frozen in liquid nitrogen and centrifuged after thawing (4°C, 15 min, 14,000 g). The supernatant was transferred to clean 1.5 ml tubes (Sarstedt), centrifuged again, and the supernatant was collected into Eppendorf tubes and stored at −80°C for further analyses. Total protein concentration was determined by the modified Lowry assay (Bio‐Rad) according to the manufacturer's protocol.