Western blots were performed as previously described [19 (link)]. Briefly, whole cell lysates or homogenized xenograft specimens were isolated using RIPA [10 mM Tris base pH 7.2, 150 mM NaCl, 1% Na-deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS)] or mTOR lysis buffers supplemented with protease inhibitors (Sigma), phosphatase inhibitors (Sigma) and phenylmethanesulfonylfluoride. Lysates were cleared by centrifugation at 14,000 rpm for 30 min at 4 °C. Protein concentrations were determined using BCA Protein Assay Reagent (Pierce, Rockford, IL) and separated by electrophoresis on sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels. Antibodies were used according to manufacturer’s recommended conditions. Molecular weight markers (Precision Plus Protein Kaleidoscope Standards, Bio-Rad, Hercules, CA) confirmed the expected size of the target proteins. Immunoblots were developed with Luminata Classico or Crescendo ECL (EMD Millipore). Blots were stripped with stripping solution (Bio-Rad) at 37 °C for 15 minutes and then reprobed with selected antibodies. Equal protein loading was confirmed with immunoblotting with antibody to GAPDH or β-actin.