Figure 3.

GAPDH limits the dephosphorylation of activated Akt by direct binding. (a) Mock or GAPDH-V5 HeLa cells were deprived of serum for 16 h and then stimulated with 200 nM insulin for the indicated time periods. Akt and Erk activation was assessed by immunoblots of phospho- or total protein. (b) Mock or GAPDH-V5 HeLa cells were treated with insulin (200 nM) for 30 min as described in panel a, lysed on ice without phosphatase inhibitors and then incubated at 30 °C for the indicated time periods. Akt activation was assessed by immunoblot and the pAkt/Akt ratio was quantified, shown in the lower panel, and normalized to time ‘0' of the Mock cells. (c) Mock or GAPDH-V5 HeLa cells were immunoblotted for the indicated proteins. (d) 293T cells were transiently co-transfected with a GAPDH-V5- and an Akt-encoding vector. After 48 h, the cells were subjected to immunoprecipitation using anti-pAkt (Ser⁴⁷³) or an IgG control. Presence of co-immunoprecipitated GAPDH was assessed by western blot. (e) HeLa cells were treated or not with 0.5 μg/ml KA for 24 h and subjected to immunoprecipitation using anti-GAPDH (left panel), anti-pAkt (Ser⁴⁷³, right panel) or an IgG control. Presence of co-immunoprecipitated Akt and GAPDH was assessed by western blot. IP, immunoprecipitation; WB, western blot. Results are representative of three independent experiments