7.4 P-AKT

The PI3K/Akt Signaling Pathway Plays an Important Regulatory Role in Many Biological Functions of Cells
The PI3K/Akt signaling pathway plays a crucial regulatory role in numerous biological functions of cells, exerting effects of inhibiting apoptosis and promoting proliferation within cells. As a downstream protein of PI3K, Akt has two key phosphorylation sites: Thr308 and Ser473.
PI3K cannot activate Akt directly. Instead, it binds to the intracellular signaling protein Akt (which contains a PH domain) and phosphoinositide-dependent kinase 1 (PDK1), inducing Akt to translocate to the cell membrane. Subsequently, it catalyzes the phosphorylation of the Thr308 site on the Akt protein, thereby partially activating Akt. However, the full exertion of Akt's functional activity also requires the phosphorylation of the Ser473 site.
In Many Cell Lines (Especially Tumor Cell Lines)
In many cell lines (particularly tumor cell lines), the inhibition of Akt function is mostly achieved by inhibiting PI3K function (e.g., using PI3K-specific inhibitors such as LY294002 and Wortmannin), with the decreased expression level of p-Akt (Ser473) serving as the main indicator.
During the Western Blotting (WB) Process, Improper Sample Handling Is the Main Cause of Problems with Phosphorylated Akt Bands
During the WB process, improper sample handling is the primary reason for issues regarding phosphorylated Akt bands.
Therefore, we must pay attention to adopting a reasonable induction method. The type, intensity (concentration), and duration of stimulation all affect the level of phosphorylation.
The phosphorylation of Akt occurs rapidly, and the signal also fades quickly. It is generally recommended to treat samples for a short period; for example, treating with insulin, Calyculin A, PDGF, etc., for 10-30 minutes can yield a strong Akt phosphorylation result.