T per reagent

Both protein samples of cerebrums and DBT cells were extracted using a protein lysis buffer called T-PER reagent (78510, Thermo Fisher Scientific, Waltham, MA, USA) and quantified by Bradford assay with PRO-Measure solution (#21011, Intron, Kirkland WA, USA). Depending on the protein size, the samples were run using SDS-PAGE electrophoresis on 10 or 12% polyacrylamide gels and transferred to the membrane. The membranes were blocked for 1 h with 30 mg/mL BSA100 (9048-46-8, LPS solution, Daejeon, Korea), diluted TBS-T buffer (04870517TBST4021, LPS solution). Primary antibodies were operated overnight at 4 °C. Following this step, the membranes were washed with TBS-T, and the secondary antibodies were operated in an identical way. Results were detected with ECL solution (XLS025-0000, Cyanagen, Bologna, Italy) and Chemi Doc (Fusion Solo, VilberLourmat, Collégien, France). The primary antibodies were diluted at 1:2500 in 5% w/v BSA, and the secondary antibodies were diluted at 1:2500 in 5% w/v skim milk. The primary and secondary antibodies information is in Table 1.

Exosomes were pelleted from CGM (10 mls) following ultracentrifugation at 100,000× g for 90 min. The exosomal pellet was re-suspended in 50 mL T-PER reagent (Thermo Scientific, Waltham, MA, USA, cat. # 78510) with the addition of protease-inhibitor cocktail (Thermo Scientific, Waltham, MA, USA, cat. # 87785). Protein concentration was determined with a bicinchoninic acid assay (Thermo ScientificWaltham, MA, USA, cat. # 23225).

Total protein was extracted from the hypothalamus, dorsal hippocampus and ventral hippocampus using T-Per reagent (ThermoFisher Scientific, Waltham MA) supplemented with Pierce Protease and Phosphatase Inhibitor Tablet, EDTA Free (ThermoFisher Scientific, Waltham MA). Similarly, total protein was extracted from the left ventricle of the heart using RIPA buffer supplemented with Pierce Protease and Phosphatase Inhibitor Tablet, EDTA Free (ThermoFisher Scientific, Waltham MA). Protein concentrations were measured using the Pierce BCA Protein Assay kit according to manufacturer’s directions (ThermoFisher Scientific, Waltham MA).

Western blot analysis was conducted to confirm the results of proteomic analysis. The heart tissues collected at 6 weeks of age were homogenized in lysis buffer (T-PER reagent; Thermo Fisher Scientific, Waltham, MA). Samples (n = 6 per group) containing 12 μg cardiac proteins were separated by 12% SDS-PAGE and electroblotted onto polyvinylidenedifluoride (PVDF) membranes. The membranes were incubated with a rabbit polyclonal antibody to peroxiredoxin 2 (PRDX2) (ab59539; Abcam, Cambridge, United Kingdom) at 1:2,000 dilution. A rabbit monoclonal antibody to GAPDH at 1:2,000 dilution (14C10; Cell Signal Technology, Danvers, MA) was used as a loading control. The protein bands were visualized by ECL plus Western blotting detection system (GE Healthcare) and Quantity One v3.0 software (Bio-Rad Laboratories) was used to quantitate the band intensities. To prevent the detection as a saturated signal, we confirmed the antibody dilution concentration and exposure time of the CCD camera in the preliminary experiments. The expression level of PRDX2 was normalized relative to the level of GAPDH protein in the same tissue sample.

Brain, retinas and RPE/choroid/sclera were dissected from wildtype and Tspo KO mice, then homogenized in cold T-PER reagent (Thermo Fisher Scientific, Paisley, UK). The tissue lysates were centrifuged at 10,000× g for 10 min. The supernatants were collected and the concentration was measured. Then, 50 µg proteins from each sample were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred into nitrocellulose membrane. The membrane was initially blocked with 5% non-fat dry milk powder in PBS, then incubated with primary antibodies and corresponding secondary antibodies respectively. Targeted protein signals were detected using the LI-COR Odyssey FC Imaging System.