(A-D) K562 cells were treated as indicated with 30 nM MTH and 1 μM RAPA and analyzed after 2, 4, 24, 48 hours by Western blotting using monoclonal antibodies against p70S6Kinase (A, C) and against the phosphorylation site of p-p70S6K Thr389 (B, D); (-) = untreated cells. (E, F) Determination of raptor protein production using specific monoclonal antibodies in untreated K562 cells and in K562 cells treated with MTH for 24 and 48 hours (when the modification of p70S6Kinase phosphorylation occurs). Western blotting was performed using antibodies against raptor protein (arrowed). G, H) Western blotting showing expression of mTOR, used as loading control in addition to Ponceau S staining. (I, L) The densitometric analysis of K562 cells were treated as indicated with 30 nM MTH and 1 μM RAPA and analyzed after 2, 4, 24, 48 hours by Western blotting using monoclonal antibodies against the phosphorylation site of p-S6RP Ser235/236. The quantitative densitometric analysis of all the autoradiograms is shown in the lower part of the panels. Normalized intensity values are expressed as the mean (black) and standard deviation (white) from three independent experiments.