Fig. 2. Silencing of RPS27L inhibits mTORC1 activity in breast cancer cells.

a, b Silencing of RPS27L inhibits the phosphorylation of mTORC1 downstream effectors. Cells were transfected with scramble control siRNA or siRNA targeting RPS27L for 48 h, followed by IB with indicated Abs. b Silencing of RPS27L inhibits the phosphorylation of mTORC1 downstream effectors triggered by serum addition after serum starvation. Cells were transfected with indicated siRNA oligos for 48 h, and then serum starved for 24 h, followed by addition of serum for indicated time periods. Cells were then harvested for IB with indicated Abs. Densitometry quantification was performed with ImageJ. The ratios of phosphorylated levels and total protein levels were shown. SS serum starvation, FBS fetal bovine serum. c Silencing of RPS27L inhibits mTORC1 activity, but does not affect mTORC2 activity in in vitro kinase activity assays. HA-tagged S6K1 or HA-tagged AKT1 was transfected into 293 cells, followed by immune-affinity purification by immunoprecipitation using bead-conjugated HA antibody. After elution with HA peptide, HA-S6K1 (left panel), or HA-AKT1 (middle panel) was added into a kinase reaction mixture containing mTOR complex, which was immunoprecipitated by anti-mTOR Ab from MB231 cells upon silencing of RPS27L for 48 h, along with siRNA control. The reaction mixture was incubated for 90 min at 30 °C with constant shaking, followed by IB with indicated Abs. The whole-cell extracts (WCE) (right panel) of MB231 cells were subjected to IB with indicated Abs. Densitometry quantification was performed with ImageJ. The ratios of phosphorylated levels and total protein levels were shown