Figure 4. CaM is required for hVps34 activity and mTOR Compex1 signaling. (A).

HeLa cells were treated with AAs as in Figure 1A, in the absence or presence of 10 μM W7. W7 was added 15 min prior to the AA stimulation. (B) HeLa cells were transfected with either 45 nM nonsilencing (NS) siRNA or an siRNA mix containing 15 nM each of CaM1, CaM2, and CaM3 siRNA, as in Figure 1A (Experimental Procedures). Following transfection, cells were treated with AAs as in Figure 1A. The hVps34 activity assay was performed on hVps34 immunoprecipitated as described (Experimental Procedures). (C) HeLa cells were treated with AAs as in Figure 1A. Extracts were prepared in the absence of Ca²⁺, and then divided into 2 aliquots, with one brought to 0.5 mM CaCl₂ and the other to 2 mM EGTA. CaM-agarose beads were added to each aliquot of extract, and the binding assays were performed as described (Vergne et al., 2003). Right panel, endogenous mTOR was immunoprecipitated from cells in the presence of either 2 mM EGTA or 0.5 mM CaCl₂.