Downregulation of mTOR, rictor and raptor in HT29 and LS174T colon cancer cells. (A) Knockdown of raptor, rictor or mTOR in HT29 (HT) and LS174T (LS). HT29 and LS174T cells were infected with recombinant lentiviruses containing the indicated shRNA. Two days post infection cells were selected for resistance to puromycin for 48 hours. Resistant cells were plated in 6 well plates and cultured in DMEM 10% FBS for 2 days. Cells were subsequently lysed in RIPA lysis buffer containing a protease inhibitor cocktail (Sigma) and 1 mM sodium orthovanadate. Proteins were quantified with the BCA protein Assay Kit (Pierce). Equal amounts of proteins (30 μg) were resolved on SDS PAGE, transferred to membranes and immunoblotted with primary antibodies against mTOR, rictor, raptor, phospho-Akt (Ser 473), Akt, phospho-S6K1 (Thr 389) or S6K1 (Cell Signalling Technology) followed by infrared secondary antibodies (LI-COR Biosciences). Bands from immunoreactive proteins were visualised by an Odyssey infrared imaging system (LI-COR Biosciences). (B) Rapamycin inhibits mTORC1 but not mTORC2 in HT29 and LS174T cells. HT29 or LS174T cells were treated with rapamycin (10 ng/ml) for the indicated times. Cells were subsequently lysed and Western Blot analysis was performed as in A. The illustrated blots are representative of three with similar findings.