The PI3K/Akt signaling pathway controls a variety of cellular functions including glucose metabolism, cell survival, proliferation, and invasiveness. Due to its role in these important processes, proper regulation is critical as constitutive activation of Akt has been observed in a variety of cancers including neuroblastoma, breast cancer, and prostate cancer. In this video, we use pan and phospho specific Akt antibodies from Cell Signaling Technology (CST) for an in-depth review the upstream signaling events that regulate Akt using confocal immunofluorescent analysis. The PI3K/Akt pathway is initiated when growth factors or cytokines signal through membrane receptors including receptor tyrosine kinases (RTKs), cytokine receptors, G-protein coupled receptors, B-cell receptors, and integrin receptors. These receptors signal through PI3K which in turn phosphorylates PIP2, converting it into PIP3. PIP3 then binds to the PH domain of Akt, anchoring it to the cell membrane where the kinase PDK1 and mTORC2 complex activates the membrane bound Akt through phosphorylation on Thr308 and Ser473 respectively. This triggers activation of downstream proteins (substrates). PI3K/Akt signaling is inactivated when the phosphatase PTEN converts PIP3 back to PIP2, leaving Akt in its inactive dephosphorylated state. Visit http://www.cellsignal.com to see antibodies for the study of Akt Signaling.