Endosomes were isolated using Trident Endosome Isolation Kit (GeneTex, GTX35192). Briefly, 3 × 107 cells from each sample were collected and washed once with cold PBS. The supernatant was removed completely, and the cell pellet was resuspended in 500 μL buffer A. The cell suspension was incubated on ice for 5–10 minutes and vortexed vigorously for 10–30 seconds, and then transferred to a filter cartridge. To remove the mitochondria, the cell suspension was centrifuged at 16,000 g for 30 seconds. The pellet was resuspended by vortexing briefly and centrifuged at 700 g for 2–3 minutes. The supernatant was carefully transferred to a fresh 1.5 mL tube and centrifuged at 16,000 g at 4°C for 30–60 minutes. The resulting supernatant was transferred to a fresh 1.5 mL tube, added with buffer B and vortexed briefly. The tube was incubated at 4°C overnight, and centrifuged at 10,000 g for 30 minutes at 4°C. After removing the supernatant, the pellet contained isolated endosomes.

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