Fig. 5.

PA alters Akt and Rictor phosphorylation, as well as mTORC2 protein-protein interactions. C2C12 myocytes were incubated overnight in the presence or absence of 100 mM EtOH. For PA treatment, cells were incubated in SFM with PA (30 µM) for 75 min. For combination experiments, EtOH pre-treated cells were incubated in SFM with PA (30 µM) for 25 min. EtOH was then added to the media for another 50 min in the continued presence of PA. Equal amounts of protein from cell lysates were analyzed via Western blotting using antibodies against the phosphorylated forms of Akt (A) and Rictor (B). (C) Rictor was immunoprecipitated from equal amounts of cell extract and then immunoblotted with antibodies as indicated. Results were quantified as bar graphs (D–F) and normalized with immunoprecipitated (IP) Rictor that was assessed by immunoblotting. The data shown are mean ± SE of 4 independent experiments consisting of 3–5 replicate samples per experiment. Groups with different letters are significantly different from one another (P< 0.05). Groups with the same letters are not significantly different. *P< 0.05 versus the control values.