Cellular Analysis Using the MILLIPLEX® MAP™ Akt/mTOR Signaling Pathway Panels

Abstract

The Akt/mTOR pathway plays a central role in a number of key cellular processes including translation, cell cycle, cellular energy homeostasis, and apoptosis. The study of this pathway is important for an understanding of signaling involved in physiological as well as several pathological states including cancer and diabetes. We have developed a Luminex®‐based multiplex assay that allows for the simultaneous detection of multiple phosphorylated Akt/mTOR pathway proteins in a single well. Using the Akt/mTOR Multiplex Panels we observed increased levels of phosphorylated IR, IGF1R, IRS1, Akt, GSK3α, GSK3β, p70S6K, and RPS6 in stimulated cells when compared to their unstimulated counterparts. Modifications in phosphorylation levels were confirmed by the treatment of cells with specific inhibitors, such as wortmannin which blocked phosphorylation of PRAS40, mTOR, TSC2, Akt, GSK3α, GSK3β, p70S6K, and RPS6, as well as with phosphatase treatment of cell lysates. The Akt/mTOR signaling panel results were more quantitative, sensitive, and required much less labor compared to Western blotting when used to analyze phosphorylated protein biomarkers in cellular lysates. Support for this research is sponsored by Millipore Corp.