AKT Kinase Activity Assay

The AKT Kinase Assay kit was purchased from the Cell Signaling (Danvers, MA, # 9840). As instructed by the provided protocol, immobilized AKT antibody was used to immune-precipitate AKT from the indicated cell extracts [48 (link)]. Immune-precipitated pellets were then incubated in the Kinase Buffer containing GSK-3 fusion protein and cold ATP. Levels of GSK-3 phosphorylation using the anti-phospho-GSK-3α/β (Ser21/9) antibody were measured by western blotting and chemiluminescent detection. All the experimental steps were carried out strictly according to the instructions of the kit. Each experiment was repeated for three times.