Rapamycin Effects on PROX1 and MTOR Signaling

Cells (2 × 10⁵) were plated in 60 mm dishes, treated with the indicated concentrations of rapamycin for 0~48 h, and then lysed using RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], and 0.5% sodium deoxycholate) containing protease and phosphatase inhibitors (Sigma-Aldrich, San Diego, CA, USA). Proteins were quantified using a Bradford Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA). For Western blot assay, 20 μg of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (GE HealthCare, Hatfield, UK), which were then treated with anti-PROX1 antibody [25 (link)], anti-phospho-MTOR (p-MTOR, ser-2448) antibody (sc-101738, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MTOR antibody (Ab-2448, Cusabio, Houston, TX, USA), anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, CA), appropriate secondary antibodies, and enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).