Figure 2.

Reversal by PP1_C of the effects of insulin on mTOR. (A) 3T3-L1 adipocytes were incubated at 37°C for 30 min in HBS without or with 10 milliunits/ml insulin before extracts were prepared and mTOR was immunoprecipitated. The beads were then suspended in 50 μl of buffer A (see legend to Fig. 1) containing 1 mM MnCl₂ and incubated at 22°C for 30 min without or with 0.5 μg of recombinant PP1_C. The beads were washed three times in buffer A (1 ml per wash) containing 500 nM microcystin-LR, then incubated for 30 min at 30°C in 50 μl of reaction mix before ³²P incorporation into PHAS-I was determined. The results are expressed as percentages of the ³²P incorporation observed with mTOR immunoprecipitated from control cells and are mean values ± SE of three experiments. (B) Other adipocytes were incubated without or with 10 milliunits/ml insulin for 30 min before mTOR was affinity purified by using the rapamycin⋅GST-FKBP12 resin. The beads were incubated at 22°C for 2 hr without or with PP1_C, then washed as described above. Proteins were eluted, subjected to SDS/PAGE, and transferred to Immobilon membranes. After an immunoblot was prepared with mTAb1, the membrane was stripped and reprobed with mTAb2. (C) The relative amounts of mTAb1 bound to mTOR were determined by laser densitometry. Mean values ± SE of three experiments are presented.