Secondary antibody conjugated to horseradish peroxidase

Proteins were extracted from subconfluent cultures of cells and then characterized by Western blot analysis. Cells were lysed in RAPI with phosphatase inhibitor cocktail, protease inhibitor cocktail, resolved on a sodium dodecyl sulphate‐polyacrylamide electrophoresis (SDS) gel and transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% non‐fat milk in phosphate buffer saline (PBS) containing 0.05% Tween‐20 (PBST) for 1 hour at room temperature and then probed with a primary antibody overnight at 4°C. After extensive washing, the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase (1:10 000, Proteintech) for 1 hour at room temperature. Blots were developed using ECL (Thermo Fisher Scientific, USA).

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 was extracted using ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Cwbio, China) containing a phosphatase inhibitor (Roche, Switzerland) and a protease inhibitor cocktail (Roche). After the protein was separated by 10% SDS-PAGE, the wet transfer method was used to transfer the isolated proteins to nitrocellulose blot (Millipore, Germany) at 200 mA for 1 h. After 1 h blocking, the blot was incubated overnight at 4°C with antibodies against proliferating cell nuclear antigen (PCNA) (1:1,000; Santa Cruz, United States). On the second day, the blot was washed and then incubated with the respective secondary antibody conjugated to horseradish peroxidase (Proteintech, United States) for 1 h at room temperature. A G-Box iChemi Chemiluminescence image capture system (Syngene, United States) was used to visualize the bands. The same blot was also probed with actin (1:10,000; Proteintech, United States) as internal controls. The experiments were repeated at least three times.