*4.3. Western Blotting*

Serum exosomes were lysed in RIPA buffer and the protein concentration was determined using the Quick Start Bradford protein assay (Pierce™ BCA Protein Assay kit, Thermo Fisher Scientific, MA, USA). The lysates (10 μg) were electrophoresed through 8–12% SDS-PAGE gels, and the separated proteins were transferred to a nitrocellulose membrane. The membrane was incubated in blocking buffer (5% milk in Tris-buffered saline with 0.05% *w*/*v* Tween-20) for 1 h at room temperature and then incubated overnight at 4 ◦C with mouse antibody against rat CD63 (1:1000, BD Pharmingen, CA, USA) and CD9 (1:1000, BD Pharmingen). Immunopositivity was detected with a horseradish peroxidase (HRP)—conjugated secondary antibodies and the Pierce enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific, MA, USA). The data were recorded and analyzed using the ChemiDoc Imaging System (Bio-Rad).

#### *4.4. Isolation of Total RNA fromEexosomes and Next-Generation RNA Sequencing*

Total RNA was isolated from serum exosomes using the SeraMir Exosome RNA Purification Column kit (System Biosciences, CA, USA). For each sample, 1 μL of the final RNA eluate was used for measurement of RNA concentration with the Agilent Bioanalyzer Small RNA Assay using the Bioanalyzer 2100 Expert instrument (Agilent Technologies, Santa Clara, CA, USA). Serum exosomal RNAs (N = 6 each group) were sent to the Exo-NGS™ (Exosomal RNA-Seq) services for next-generation RNA sequencing (System Biosciences, CA, USA) using small RNA libraries. Next-generation RNA sequencing was performed on an Illumina NextSeq instrument (Illumina, CA, USA) with 1 × 75 bp single-end reads at an approximate depth of 10–15 million reads per sample.
