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VEGF secretion was measured in RCC cells using Human VEGF ELISA kits (RayBiotech, Inc., Norcross, GA). Cells were plated in 6-well plates for 24 h, and then treated with physapubescin both under normoxia and hypoxia conditions for 24 h. Supernatants were collected and VEGF protein levels were determined by ELISA according to the manufacturer’s instructions. Absorbance was measured using a microplate reader (Bio-Rad, Hercules, CA, USA).
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