Figure 3

Quantifying phosphorylation dependent interactions between Bad and 14-3-3 isoforms. (a) CMV-driven expression constructs are depicted as bar graphs. Murine Bad, mutated Bad-S136A and murine 14-3-3 isoforms ε and ζ were fused to N-terminal or C-terminal TEV fragments as indicated. N-terminal and C-terminal TEV fusions contain a Flag-tag or an HA-tag, respectively. The constitutive form of murine Akt-1 (myr-Akt-Δ4-129-HA) lacks the PH domain, but contains an N-terminal myristoylation signal and a C-terminal HA-tag. (b) Western blot analysis of expression constructs shown in (a). Calculated protein sizes are: N-TEV-Bad, 41.0 kDa; N-TEV-Bad-S136A, 41.0 kDa; Const-Akt, 43.4 kDa; 14-3-3ζ-C-TEV, 42.8 kDa; 14-3-3ε-C-TEV, 44.2 kDa. (c) Principle of TEV fragment fusion proteins used to monitor the phosphorylation dependent interaction of Bad and 14-3-3. When Akt-1 kinase activity is present, Bad becomes phosphorylated enabling 14-3-3 to bind and resulting in reconstitution of the TEV protease activity. Const-Akt, constitutively active form of murine Akt-1. (d) Luciferase reporter assay quantifying the regulated interaction of murine Bad and 14-3-3 isoforms in NIH-3T3 cells. Cells were transfected with the indicated constructs. Mock treated cells were transfected with pcDNA3 only. Const-Akt, constitutively active form of murine Akt-1. RLUs, relative luciferase units, n = 4. (e) Renilla luciferase readings in NIH-3T3 cells show that only phosphorylated Bad influences the cellular state. Measurements were obtained from the same experiment as shown in (d). NIH-3T3 cells were transfected with the indicated constructs and the ratio of the mean value of the Renilla luciferase readings over the number of seeded cells per well (30000) were plotted.