Figure 5

Insulin-induced phosphorylation of Akt and GSK3β was inhibited by E2 protein. After being transfected with FLAG-CMV2 or FLAG-E2 for 24 hours and a serum-free incubation 16 hours, cells were subjected to a insulin stimulation for an indicated time period (0, 15, 30 and 60 minutes) followed by western blotting with indicated antibodies to reveal a time-dependent increase of phosphorylation of Akt (A) and GSK3β (C). Furthermore, transfected or untransfected cells were treated insulin for 30 minutes and then subjected to western blotting to detect Ser473 and Thr308 phosphorylation of Akt (B) serine phosphorylation of GSK3β (D). Results suggested that an expression of E2 inhibited insulin-induced Thr308 phosphorylation of Akt and GSK3β phosphorylation.