The indicated OS cells at 80% confluence were incubated in serum-free DMEM for three days. The CM were collected, filtered, and then added 4 volumes of cold acetone. After mix and keep at -20 °C overnight. Then spin at maximum speed for 15 min at 4 °C and discharge supernatant and retain the pellet. Dry samples under vacuum and resuspend samples in a minimal volume of RIPA buffer. The expression of indicated protein was detected by western blot.

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