Figure 6

Bad/14-3-3 split-TEV assays monitoring the effects of transiently activated endogenous Akt-1. (a) Western blot analyzing the stimulus-dependent activated form of Akt-1. NIH-3T3 cells were treated either in 1%, 5%, 10% or 1% FCS and PDGF-BB (P). The phosphorylation of Akt-1 at residue T473 was analyzed after 1 h, 6 h and 24 h after treatment using an α-P-Akt antibody. Loading was controlled using an α-Akt antibody. (b) Relative quantification of the western blot shown in (a) showing the transient activation of endogenous Akt-1 1 h after stimulation of NIH-3T3 cells. (c) Split-TEV assay using Bad-N-TEV and 14-3-3ζ-C-TEV constructs to measure endogenous activated Akt-1. NIH-3T3 cells were treated as in (a). Analysis was performed 6 h after treatment. RLUs, relative luciferase units, n = 4. (d) Renilla luciferase readings in NIH-3T3 cells. Measurements were obtained from the same experiment as shown in (c). NIH-3T3 cells were transfected with Bad-N-TEV and 14-3-3ζ-C-TEV constructs. The mean value of the Renilla luciferase readings were given as a ratio of the values versus the number of seeded cells per well (30000).