Effect of MPA treatment on the mTOR pathway. 32D cells were treated with 2 μM MPA for the indicated time points. Total cell lysate (100 μg) was used for phospho-S6 kinase detection (A, upper panel); the membrane was probed with antibody against total S6 kinase (A, lower panel) and 4E-BP1 (B). FL5.12 cells were treated with 2 μM MPA alone or MPA plus 100 μM guanosine and were harvested at indicated time points; 40 μg protein was analyzed for phospho-S6K and total S6K (C). 32D cells were treated with 1 or 2 μM MPA in the absence or presence of 100 μM adenine or 100 μM guanosine for 4.5 hours; 40 μg lysate was analyzed for phospho-S6K and total S6K (D).