Total protein in the lung tissues of mice was extracted by cell lysis buffer kits (P0013B, Beyotime) for the measurement of protein levels of TOP2A and VEGF. The mice serum was appended with extraction buffer and centrifuged at 4℃ and 14,000 rpm for 10 min. The supernatant was used for the assessment of protein levels of exosome marker proteins (CD63, HSP70 and TSG101). The BCA kits (P0010, Beyotime) were used to determine the protein concentrations: the proteins were conducted with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis for 2 h and transferred onto membranes, which were blocked with 5% skim milk powder for 2 h and supplemented with 4 mL primary antibodies TOP2A (1: 100), VEGF (1: 1000), β-actin (1: 500, all from Santa Cruz Biotechnology Inc, CA, USA), CD63 (1: 800), HSP70 (1: 1000) and TSG101 (1: 3000, all from Abcam Inc., Cambridge, MA, USA) and incubated at 4℃ overnight. Subsequently, the membranes were added with 4 mL secondary antibody goat anti-rabbit immunoglobulin G/HRP and incubated for 1 h, then exposed and developed. With β-actin as the loading control, the gray values were analyzed by the Image Lab software and protein expression was calculated. The measurement of protein expression of TOP2A, VEGF, CD63, HSP70 and TSG101 in ECs of mice was same as above.

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