Ab193349

SDS-PAGE and immunoblotting were performed as we previously described.⁶⁴ (link) Briefly, 8% SDS-acrylamide gel was used for detecting APP (1:1,000; ab12269; Abcam). 10% SDS-acrylamide gel was used for detecting BDNF (1:1,000; ab223354; Abcam), Rela (1:1,000; 8242; Cell Signaling Technology), and ApoE (1:1,000; ab183597; Abcam). 12% SDS-acrylamide gel was used for detecting CD9 (1:2,000; ab92726; Abcam), CD63 (1:1,000; ab193349; Abcam), CD81 (1:1,000; 10037; CST), neurogranin (1:1,000; ab217672; Abcam), VILIP-1 (1:1,000; ab187631; Abcam), and Aβ (1:1,000; 15126; CST). GAPDH (1:2,000; 2118; CST) and α-tubulin (1:1,000; 2144; CST) were used as the internal control. For densitometry, the ChemiDoc XRS+ Imaging System (Bio-Rad, Hercules, CA, USA) was used. Measurement of mean pixel density of each band was conducted using Quantity One software (Bio-Rad).

Cells were plated in a 100-mm culture plate with 8 mL of media. For immunoblotting analysis, 20 μg of protein was resolved with 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel and transferred to polyvinylidene difluoride membranes (GE Healthcare Life science, Pittsburgh, PA, USA). The membranes were probed with anti-CD9 antibody (EPR2949, rabbit monoclonal, ab92726, Abcam, Cambridge, UK), anti-CD63 antibody (MX-49.129.5, mouse monoclonal, ab193349, Abcam), and anti-beta-actin antibody (C4, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies used were horseradish peroxidase (HRP)-goat anti-rabbit immunoglobulin (IgG) antibody, HRP-conjugated goat anti-mouse IgG F(ab')₂ (both from Enzo Life Sciences, Farmingdale, NY, USA), and HRP-conjugated goat anti-mouse IgG (H+L) antibody (#1706516, Bio-Rad Laboratories, Hercules, CA, USA). The images were detected using an ECL chemiluminescent substrate (GE Healthcare Life Science) and analyzed using a LAS-3000 imager (Fujifilm, Japan).

Total protein samples for western blot analysis were extracted from cultured NRNs or the left ventricle and hippocampi of the mice after they were anaesthetized with sodium pentobarbital (100 mg/kg, i.p.). Mouse death was then confirmed by exsanguination according to a previously described method [31 (link)]. Hippocampi for primary cell culture were collected from neonatal Sprague-Dawley (SD) rats after the administration of 20% isoflurane and confirmation of death by cervical dislocation. Anti-SNAP-25 (1:1000, ab5666, Abcam, MA, USA), anti-VAMP-2 (1:10000, 104,211, Synaptic Systems, Gottingen, Germany) and anti-Syntaxin-1A (1:5000, 100,111, Synaptic Systems, Gottingen, Germany), anti-Munc-18 (1:1000, 116,002, Synaptic Systems, Gottingen, Germany), anti-CD63 (1:1000, ab193349, Abcam, MA, USA),were used as primary antibodies. β-actin (1:1000, G8795, Sigma, Saint Louis, MO, USA) was selected as an internal control. The blots were detected with an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The final results were expressed as fold changes compared with the control values.

BMSCs or exosomes were lysed by RIPA buffer (Beyotime, Shanghai, China). The isolated proteins were then further transferred to a PVDF membrane (Millipore), which was further blocked by non-fat milk before incubation with primary antibodies, including anti-CD63 (Abcam, ab193349, 1:1000 dilution), anti-TSG101 (Abcam, ab125011, 1:1000 dilution), anti-RUNX2 (Abcam, ab236629, 1:1000 dilution), anti-Collagen I (Abcam, ab260043, 1:1000 dilution), anti-OPN (Abcam, ab283656, 1:1000 dilution) and anti-GAPDH (ABclonal, AC002, 1:1000 dilution) overnight, and corresponding secondary antibodies for 1 h. Finally, the membrane was washed with PBST and reacted with a chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA). The band was imaged by a Tanon 5200 Multi Scanning System. This experiment was performed in triplicate.

The purified sEVs and A549 cells were lysed with 1 × RIPA buffer and cocktails and centrifuged at 13,000×g for 15 min to obtain the supernatant. Protein concentrations were detected with BCA Protein Assay according to the manufacturer’s protocols. sEVs markers CD9 (Abcam, ab92726), CD63 (Abcam, ab193349), TSG101 (Abcam, ab83), and sEVs non-markers calnexin (Abcam, ab22595), GM130 (Abcam, ab52649), Lamin B1 (Abcam, ab16048), and GRP94 (Proteintech, 14700-1-AP) were measured to validate the expressions of sEVs as described in prior studies^{52 (link),53 (link)}.

For phenotypic characterization, MSCs and MSC-EVs were lysed via lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM Na₃VO₄, and 1 mM PMSF). Equal amount of total proteins from samples (20 μg or 40 μg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and detected via Western blot analysis. The primary antibodies used in the study included the following: CD63 (0.5 μg/ml; ab193349, Abcam, Cambridge, MA), CD81 (1:1000; sc-166029, Santa Cruz Biotechnology, Santa Cruz, CA), CD105 (1:1000; ab169545, Abcam), CD44 (1:5000; ab157107, Abcam), GM130 (1:5000; ab52649, Abcam), and Calnexin (1:5000; ab133615, Abcam). After washing, membranes were incubated with secondary antibodies conjugated to horseradish peroxidase. Goat anti-Rabbit lgG (H+L) HRP Conjugated (1:5000, GAR0072, Multi Sciences, Hangzhou, China) and goat anti-Mouse lgG (H+L) HRP Conjugated (1:5000, GAM0072, Multi Sciences, Hangzhou, China) were used as Secondary antibodies. The signals were detected via enzyme-linked chemiluminescence using the EZ-ECL kit (Biological Industries, Kibbutz Beit-Haemek, Israel).

Cultured cells were collected and lysed in lysis buffer containing a proteinase inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF, Cell Signaling Technology, Danvers, MA, USA). Equal amounts of proteins were separated on 8–12% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and then were transferred to PVDF membranes (Millipore, Boston, MA). The membranes were then blocked with 5% nonfat milk for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. After that, blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and were visualized with enhanced chemiluminescence as previously described.^{36 (link)} The antibodies used in this study were as follows: rabbit anti-integrin alpha 2 (ab133557, Abcam), rabbit anti-integrin beta 1 (#9699, Cell Signaling Technology), mouse anti-GAPDH (MB001, Bioworld Technology), rabbit anti-HSP70 (BS6446, Bioworld Technology), rabbit anti-CD9 (#13174, Cell Signaling Technology), mouse anti-CD63 (ab193349, Abcam), rabbit ant-Tsg 101 mAb (E303, Bioworld Technology), HRP-conjugated goat anti-rabbit antibody (#7074, Cell Signaling Technology), and HRP-conjugated goat anti-mouse antibody (BS12478, Bioworld Technology).