Figure 6.

P2Y₁₂ contributes to mTORC1 activity through a PI3 kinase–independent pathway. Washed platelets were incubated with vehicle (0.2% DMSO) or BIM I (10 μmol L⁻¹) in the absence/presence of SQ 22536 (100 μmol L⁻¹), H89 (10 μmol L⁻¹), or R59949 (1 μmol L⁻¹) for 15 min before stimulation with thrombin (0.2 U mL⁻¹) (A) or thrombin (0.2 U mL⁻¹) and ADP (10 μmol L⁻¹), epinephrine (10 μmol L⁻¹), or IGF-1(100 nmol L⁻¹) for 15 min (B). Blots were performed on whole cell lysates with the exception of p70S6K, which was immunoprecipitated from RIPA lysates. Membranes were reprobed for α-tubulin as a loading control. The bar graphs depict quantification of pTSC2 at Ser939 and Thr1462 (ratio phosphorylated/loading control) and pp70S6K at Thr389 (ratio phosphorylated/total) expressed as a percentage of the maximal signal induced by thrombin in control conditions. **P < 0.01, indicating a significant difference from vehicle control(A, B). In order to assess whether significant rescue of the effect of BIM-1 on thrombin stimulation was achieved sample phosphorylation was compared to phosphorylation in thrombin + BIM-1–treated samples. †P < 0.05, ††P < 0.01, indicating a significant difference from thrombin + BIM-1–treated samples (A, B). Results are expressed as mean ± SE.