Figure 5.  .

Cholesterol depletion disrupts PA- and insulin-induced TOR signaling. (A) R28 cells were pretreated with MβCD (1%, 1 hour) and subsequently treated with insulin (10 nM, 15 minutes). Western blots were used to assess the phosphorylation state of p70 S6K and Erk 1/2. (B) The phosphorylation state of p70 S6K also was assessed after cholesterol depletion (MβCD: 1%, 1 hour) before PA treatment (50 μM, 15 minutes). Blots are representative of at least 3 independently performed experiments. (C) In a similar manner, R28 cells were pretreated with nystatin (50 μg/mL, 30 minutes) and treated subsequently with insulin or PA. A representative Western blot from a single n = 3 experiment is shown demonstrating the phosphorylation state of p70 S6K and Erk 1/2. (D) R28 cells were pretreated again with MβCD and treated subsequently with insulin or PA with or without repleting cells with cholesterol (100 μM, 30 minutes). Western blots were used to assess p70 levels and the phosphorylation state of p70 S6K. Representative blots from at least three independently performed experiments are shown. (E) A reconstitution kinase assay on immunoprecipated mTOR was performed by assessing the ability to transfer radiolabeled ATP to recombinant 4EBP1 after insulin or PA treatment in the presence of absence of MβCD. Representative radiograph of n = 3 separate experiments.