Figure 2.

Interference with the PeBoW complex regulates effectors of mTORC1. (A and B) HEK293 cells were transfected with 0.5 µg of HA-S6K1 DNA; 24 h later, cells were transfected with scrambled siRNA or siRNA against BOP1 (20 nM) as indicated. After 48 h, cells were starved overnight of serum and in some cases, subsequently starved of amino acids (1 h). Some cells were then stimulated with insulin (100 nM; 30 min). Cell lysates were subjected to western blot. (C) Cells were cultured in complete medium and transfected with scrambled siRNA or siRNA against BOP1, WDR12 or PES1 (20 nM). After 72 h, cells were lysed and samples subjected to western blot. (D) HEK293 cells were treated with actinomycin D (20 ng/ml) for different times. Cells were then lysed and samples subjected to western analysis. (E) T-REx cells expressing BOP1Δ were cultured in complete medium with/without doxycycline for 48 h. The cells were treated with 1 µM PP242, 100 nM rapamycin, 1 µM AZD6244 or 10 µM PF-4708671 for 1 h. Cells were lysed and 20 µg of lysate proteins were used for western blot analysis.