Figure 5

Determination of mTOR Complex 1 and mTOR Complex 2 regulation of VEGF-induced Akt activation in human EC. Panel A: Human EC were serum starved for one hour and either untreated (control) or treated with VEGF (100 nM, 5 minutes) with or without pretreatment (1 hour) with 100 nM MNTX or 100 nM temsirolimus. EC were then solublized in IP buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl₂, 1% Nonidet P-40 (NP-40), 0.2 mM phenylmethylsulfonyl fluoride, 1:250 dilution of Calbiochem protease inhibitor mixture 3) and immunoprecipitated with either anti-Raptor (mTOR Complex 1 component) or anti-Rictor (mTOR Complex 2 component) antibody. The immunoprecipitated material was run on SDS-PAGE and immunoblotted with anti-mTOR (a, d), anti-FKBP12 (mTOR Complex 1 component and direct target of temsirolimus) (b), anti-Raptor (c), anti-SIN1 (mTOR Complex 2 component) (e) or anti-Rictor (f) antibody. Panel B: Human EC were serum starved for one hour and either untreated (control) or treated with VEGF (100 nM, 5 minutes) with or without pretreatment (1 hour) with LY294002 (PI3 kinase inhibitor, 10 μM) or pretreated for 48 hours with Src or Rictor (mTOR Complex 2 component) siRNA. EC lysates were obtained, run on SDS-PAGE and immunoblotted with anti-pSer⁴⁷³Akt (a), anti-pThr³⁰⁸Akt (b), or anti-AKT (c) antibody.