Figure 3.

Time courses of mTOR phosphorylation and activation in response to insulin. 3T3-L1 adipocytes were incubated in HBS or in HBS supplemented with ³²P_i (0.5 mCi/ml) for 3 hr, then homogenized. Insulin (10 milliunits/ml) was added at the appropriate time prior to homogenization to give the treatment times indicated. mTOR activity was measured after immunoprecipitation with mTAb2. [³²P]mTOR was affinity purified from extracts by using rapamycin⋅GST-FKBP12 beads, and samples were subjected to SDS/PAGE before proteins were electrophoretically transferred to Immobilon P (Millipore) membranes. An autoradiogram was prepared to allow measurements of the relative amounts of ³²P-labeled mTOR (see Inset for example). An immunoblot was then prepared with mTAb2 to estimate the amounts of the mTOR protein present. mTOR activity was measured after immunoprecipitation with mTAb2 from extracts of cells incubated without ³²P. Samples of these extracts were subjected to SDS/PAGE and blotted with mTAb1. The relative amounts of mTAb1 reactivity (▵) were determined by laser densitometry and are expressed as a percentage of control. mTOR activities (•) and ³²P contents (▴) are expressed as percentages of the respective maximal levels. The results presented are mean values ± SE from three experiments.