FIGURE 4.

CSE induces Lys-48-linked ubiquitination of Akt. A, NHLFs were transfected with the Myr Akt/Myc-His plasmid together with the wild-type or lysine mutant HA-tagged Ubi plasmid (WT Ubi-HA, wild-type ubiquitin; KO Ubi-HA, all lysine residues replaced with arginine; K48 Ubi-HA, all lysine residues except Lys-48 replaced with arginine; K48R Ubi-HA, Lys-48R replaced with arginine). 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. Myc denotes exogenously introduced Myc-His-tagged Akt. GAPDH was used as an input control. B, quantitative results (ubiquitinated Akt/Myc).