Sodium dodecyl sulfate polyacrylamide gel electrophoresis

The cell precipitates and uEVs pellets were dissolved in RIPA buffer (Bioss, China), phenylmethanesulfonyl fluoride (PMSF) (Solarbio, China), and phosphatase inhibitor cocktail II (MedChemExpress, USA) were added, and the protein supernatant was collected after centrifugation. The extraction protein concentration was determined with a bicinchoninic acid protein assay kit (Servicebio, China). Equal amounts of proteins were separated via 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Vazyme, China). The proteins were subsequently transferred to 0.2 µm polyvinylidene difluoride membranes (Millipore, USA), which were blocked with 5% bovine serum albumin for 1 h and then incubated with primary antibodies overnight at 4 °C. The primary antibodies used were rabbit anti-CD63 (Abcam/ab92726, USA), rabbit anti-TSG101 (Abcam/ab2788, USA), rabbit anti-calnexin (Abcam/ab22595, USA), rabbit anti-NDUFS1 (ABclonal/A21192, China), and rabbit anti-GAPDH (Proteintech/10494–1-AP, China). Afterward, the membranes were incubated with a secondary antibody conjugated to horseradish peroxidase (Proteintech, China) for 1 h at room temperature and then visualized with a chemiluminescence imaging system (General Electric, USA).

Total protein extracted from cells was placed in radioimmunoprecipitation assay buffer (Vazyme Biotech Co., Ltd., Nanjing, China) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland), and protein concentrations were measured with the protein BCA assay kit (Invitrogen; Thermo Fisher Scientific). An equal amount of total protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Vazyme Biotech Co., Ltd.) and transferred to polyvinylidene fluoride membrane (Invitrogen; Thermo Fisher Scientific). After blocking with 5% nonfat dried milk, the membrane was incubated with primary antibody against AFF4 (1:1000, Cell Signaling Technology, MA, USA) and β-action (1:5000, Cell Signaling Technology), followed by horseradish peroxidase-conjugated secondary antibody. Protein detection was visualized with the enhanced chemiluminescent detection reagent (Pierce; Thermo Fisher Scientific) in LI-COR imaging system (LI-COR Biosciences, NE, USA). Protein expression levels were quantified by densitometry using Image J software (National Institutes of Health, Bethesda, MD, USA) and normalized to β-action.