FIGURE 3.

CSE induces preferential ubiquitination of active Akt. A, to determine preferential ubiquitination of active Akt, NHLFs were transfected with the Myc-His-tagged wild-type Akt (WT Akt/Myc-His), constitutively active myristoylated Akt (Myr Akt/Myc-His), or inactive Akt (T308A/S473A double mutant, DM Akt/Myc-His) plasmid together with the hemagglutinin-tagged ubiquitin (Ubi-HA) plasmid. Twenty four hours after transfection, NHLFs were exposed to 8% CSE for 8 h in the presence of 10 μm MG132. Ubiquitinated Akt was detected by His tag pulldown and Western blot (WB). Endo and Exo denote phosphorylated forms of endogenous and exogenously introduced Akt, respectively. GAPDH was used as an input control. B, quantitative results (ubiquitinated Akt/GAPDH).