Comprehensive Protein Extraction and Analysis

Whole Cell Lysis Assay kit (Keygen, KGP2100) was used to extract proteins from cells and small extracellular vesicles. Total protein concentration was analyzed using the BCA Protein Quantitation Assay kit (Keygen, KGPBCA). Western blotting was performed as previously described [19 (link)]. Antibodies used were as follows: TSG101 (1 : 1000, AF8259, Beyotime), CD63 (1 : 1000, AF1471, Beyotime), CD81 (1 : 1000, AF2428, Beyotime), P21 (1 : 1000, 10355-1-AP, Proteintech), BAX (1 : 1000, 50599-2-Ig, Proteintech), Bcl-2 (1 : 1000, 26593-1-AP, Proteintech), phosphorylated-AKT (p-AKT) (1 : 1000, 28731-1-AP, Proteintech), AKT (1 : 1000, AF1789, Beyotime), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1 : 1000, 10494-1-AP, Proteintech), and beta-actin (β-actin, 1 : 1000, 20536-1-AP, Proteintech). The bands were visualized by using enhanced chemiluminescence (Vazyme, E412-01) and analyzed with a gel documentation system. ImageJ was used for gray analysis of strips.