Pim2 regulates mTOR-C1 pathway through modulating TSC2 phosphorylation. (A) Pim inhibition leads to reduced phosphorylation on TSC2 in MM cells. KMS-11 and KMS-26 cells were treated with LGB321 (0, 0.33, and 1.0 μM) for 2 hours; TSC2 was immunoprecipitated and analyzed for phosphorylation status by a specific p-Akt substrate antibody (Ab). (B) Pim2 regulates TSC2 phosphorylation and mTOR-C1 pathway activity. Pim2 was transfected into 293A cells, with green fluorescent protein (GFP) as transfection control. After 48 hours, cells were treated with either dimethylsulfoxide (DMSO) or LGB321 (1.0 μM) for 2 hours. TSC2 phosphorylation status was examined by immunoblotting with p-Akt substrate Ab following IP of TSC2; mTOR-C1 pathway activity was examined by immunoblotting with p-S6RP Ab. (C) TSC2 knockout was generated from 293A cells by ZFN technology. (D) Pim2 failed to modulate mTOR-C1 pathway in the absence of TSC2 in 293A cells. Pim2 was transfected into 293A cells with various TSC2 background (TSC2^+/+, TSC2^+/−, TSC2^−/−) and then treated with either DMSO or LGB321 (1.0 μM) for 2 hours. mTOR-C1 pathway activity was examined by immunoblotting for p-P70S6K (T389) and p-S6RP (S235/236).