Expression of exosomal surface markers on the ExoQuick-isolated exosomes was detected by western blotting, in triplicate. Serum samples were used as a negative control. Exosome samples from the blood of CLP and sham mice were lysed, and the total proteins were separated by polyacrylamide gel electrophoresis (PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in PBS/Tween-20 and incubated with primary antibodies against CD9 (cat # ab92726, 1:1000; Abcam, Cambridge, MA, USA), TSG101 (cat # ab30871, 1:1000; Abcam), Flotillin-1 (cat # 18634, 1:1000; Cell Signaling Technology, Danvers, MA, USA), CD81 (cat # ab109201, 1:1000; Abcam), and Calnexin (cat # ab22595, 1:1000; Abcam) at 4°C overnight. The PVDF membranes were washed with 0.1% TBS/Tween 20 for 10 min, three times at room temperature and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (cat # NA931; GE Healthcare Amersham, Piscataway, NJ, USA) for 2 h at room temperature, and the detected proteins were quantified using a FluorChem SP imaging system (Alpha Innotech, San Leandro, CA, USA).

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