Figure 4

Comparison of endogenous versus exogenous Akt and Bad protein levels. (a, c) Western blot analyses to determine the relative amounts of endogenous protein levels of Akt (55.7 kDa) and Bad (22.1 kDa) versus the corresponding overexpressed variants. CMV-driven expression constructs coding for HA-tagged const-Akt (43.4 kDa) and the Flag-tagged N-TEV-Bad (41.0 kDa) and N-TEV-Bad-S136A (41.0 kDa) proteins were transfected in NIH-3T3 cells cultured in 6-well dishes at DNA concentrations adjusted to the amount used for the 96-well-luciferase assays. The upper panel shows that the endogenous Akt and Bad proteins as well as the exogenous fusion constructs can be simultaneously detected with α-Akt (a) and α-Bad antibodies (c), respectively. The lower panel probed with α-HA (a) and α-Flag (c) antibodies verifies the additional bands obtained with α-Akt (a) and α-Bad (c) as the exogenously introduced const-Akt, N-TEV-Bad and N-TEV-Bad-S136A proteins. (b) Relative quantification of the band intensities obtained in (a) reveals that the level of const-Akt protein is approximately 1.5 fold higher compared to endogenous Akt. (d) Relative quantification of the band intensities obtained in (c) reveals that the levels of the N-TEV-Bad and N-TEV-Bad-S136A proteins are between 3 and 2.5 fold higher compared to endogenous Bad.