Ab88388

After RIPA cleavage, we extracted total protein and measured with BCA method. After quantitative denaturation, 60 μg proteins were loaded via SDS-PAGE (with a constant voltage of 110 V) and transferred onto nitrocellulose membranes (with a constant current of 300 mA), then the membrane containing proteins was blocked with 5% BSA. The first incubation and second incubation were carried out according to the operation steps. The expression of the protein was expressed by the gray value. Primary antibodies list: CD63 (ab134045, Abcam), Tsg101 (ab125011, Abcam), Alix (ab88388, Abcam).

Cells or exosomes were harvested and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% Na deoxycholate, 0.1% SDS, 1 mMEDTA) supplemented with complete protease inhibitor cocktail (Sigma-Aldrich). Lysates were centrifuged at 12,000 g for 30 min at 4°C to remove insoluble material, after which protein concentrations were determined using a Bradford assay kit using bovine serum albumin (BSA) as a standard according to the manufacturer's instructions. Proteins were resolved on SDS-polyacrylamide gels, and then transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% (w/v) fat free milk, the membrane was stained with the corresponding primary antibodies followed by incubation with the appropriate secondary HRP-conjugated antibodies. The membrane was incubated with the following primary antibodies: anti-CD63 antibody (Abcam, ab134045), anti-TSG101 antibody (Abcam, ab125011), anti-ALIX antibody (Abcam, ab88388) and anti-CAV1 antibody (Abcam, ab2910), anti-ARF6 antibody (Abcam, ab131261), anti-Rac1 antibody (Abcam, ab155938), anti-CLTC antibody (Abcam, ab21679), anti-GAPDH (Cell signaling).

EVs were isolated from ADSCs using the total EV kit (Invitrogen). The ADSCs were incubated in the FBS-free medium for 48 h to collect supernatant. The supernatant was centrifuged at 2000 g at 4°C for 30 min to remove cells and debris. Next, the supernatant was centrifuged at a 100 kDa ultrafiltration device at 4000 g at room temperature for 15 min to partially enrich EVs, which was mixed with 0.5-fold volume of total EV isolation reagent. The mixture was incubated at 4°C overnight and centrifuged at 10000 g at 4°C for 60 min the next day. Precipitates were the isolated EVs, resuspended in PBS, or stored at -80°C.
In addition, the protein levels of such EV-related markers as CD9 (ab236630, 1 : 1000, Abcam), CD63 (ab216130, 1 : 1000, Abcam), ALIX (ab88388, 1 : 1000, Abcam), and calnexin (ab133615, 1 : 5000, Abcam) were measured by Western blot analysis.
The morphological characteristics of EVs were observed under the transmission electron microscope (TEM) (Hitachi H7650, Tokyo, Japan) [34 (link)]. Particle size distribution of EVs was evaluated under a NanoSight nanoparticle tracking analyzer (NTA, Malvern, Marvin, UK) [35 (link)].

Primary CECs were isolated from cerebral microvessels of healthy male adult rats (2–3 months of age) and aged-DM rats (15 months of age, 2 months of DM) according to published method (Teng et al., 2008 (link)). We have demonstrated that the purity of primary endothelial cells is above 90% (Teng et al., 2008 (link)). The sEVs were isolated from culture medium of passages 1 to 2 CECs using a ultracentrifugation method according to published protocol (Li et al., 2021 (link)). The size and particle number of sEVs were characterized by a nanoparticle tracking analyzer (NTA, NanoSight N300 System, Merkel Technologies, Israel). sEV morphology was examined by means of transmission electron microscopy (TEM, JEM-1500Flash, JEOL). Total proteins extracted from isolated sEVs were used for Western blot analysis for the identification of EV marker proteins. The following primary antibodies were used: CD63 (ab68418, 1:1,000, Abcam), Alix (ab88388, 1:1,000, Abcam), CD31 (ab124432, 1:1,000, Abcam), and Calnexin (ab22595, 1:1,000, Abcam).

Total cell protein was extracted with RIPA lysate, and the protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) in a microplate reader. After denaturation for 10 min with the addition of loading buffer, 50 μg of protein samples were subjected to SDS-PAGE and transferred onto PVDF membranes. The membrane was blocked with blocking solution (5% nonfat dry milk) for 2 h and subsequently washed three times using TBST. Specific primary and secondary antibodies were next added separately, followed by incubation on a shaker. ImageJ software was applied to detect and analyze the gray values of protein bands on the membrane. The following primary antibodies: β-actin (1 : 1000, Abcam, ab8227, Cambridge, UK), NGF (1 : 1000, ab52918, Abcam), TrkA (1 : 1000, ab109010, Abcam), PI3K (1 : 1000, ab32089, Abcam), p-AKT (phospho T308) (1 : 500, ab38449, Abcam), AKT (1 : 1000, ab8805, Abcam), mTOR (1 : 1000, ab109268, Abcam), p-mTOR (phospho S2481) (1 : 1000, ab137133, Abcam), CD63 (1 : 1000, ab134045, Abcam), TSG101 (1 : 1000, ab125011, Abcam), ALIX (1 : 500, ab88388, Abcam), CD9 antibody (1 : 2000, ab92726, Abcam), TRPV1 (1 : 1000, ab6166, Abcam).

The total protein extracted from the cell lysate using RIPA buffer (Beyotime, Shanghai, China) was quantified by a BCA assay kit (Beyotime). After 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the separated proteins were electrotransferred onto the polyvinylidene fluoride (PVDF) membrane (Millipore, Boston, MA). Subsequently, the membrane was blocked with 5% skimmed milk and probed with the following primary antibodies from Abcam (Cambridge, UK) rabbit anti-α-SMA (ab32575, 1:2500), rabbit anti-collagen I (ab34710, 1:2500), rabbit anti-SOX4 (ab80261, 1:1500), rabbit anti-DKK1 (ab93017, 1:1500), rabbit anti-β-actin (ab8226, 1:5000), rabbit anti-CD63 (ab134045, 1:1000), rabbit anti-CD81 (ab109201, 1:5000), rabbit anti-CD9 (ab92726, 1:1000), rabbit anti-Alix (ab88388, 1:1000), rabbit anti-TSG101 (ab125011, 1:1000), and rabbit anti-calnexin (ab92573, 1:20000) at 4 °C in the dark. Subsequently, the membrane was re-probed with horseradish peroxidase (HRP)-combined immunoglobulin G (IgG) antibodies (ab6721, 1:5000, Abcam) for 2 h followed by enhanced chemiluminescence (BB-3501, Ameshame, UK) for immunodetection. The protein bands were imaged and analyzed by the BIO-RAD imaging system (California).