Figure 4

Generation of 39M1-81 cells expressing a conditionally active form of Raf-1 protein kinase. (A) Schematic representation of the modular structure of Raf-1 and ΔRaf-1:ER. (B) Expression of ΔRaf-1:ER in 39M1-81 cells. Whole cell extracts of ΔRaf-1:ER expressing 39M1-81 cells were prepared and analyzed by immunoblotting using an antibody directed against the murine estrogen receptor. As a control, 39M1-81 cells were analyzed expressing the selection marker puromycin acetyltransferase (pac). (C) Upper panel: 39M1-81-ΔRaf-1:ER cells and 39M1-81pac cells were stimulated with 4OHT (100 nM) for 2 hours. Nuclear extracts were prepared and subjected to Western blot analysis. The blot was incubated with an antibody directed against Egr-1. Middle panel: Sustained synthesis of Egr-1 in 4OHT-treated 39M1-81-ΔRaf-1:ER cells. 39M1-81-ΔRaf-1:ER cells were stimulated with 4OHT (100 nM) for 2, 4, 6, or 8 hours as indicated. Nuclear extracts were prepared and subjected to Western blot analysis. The blot was incubated with an antibody directed against Egr-1. Lower panel: 39M1-81-ΔRaf-1:ER cells were stimulated for 2 hours with different concentrations of 4OHT as indicated. For comparison, cells were stimulated with carbachol (100 μM). Egr-1 expression was visualized by Western blotting using an anti-Egr-1 antibody. (D) The newly synthesized Egr-1 protein in 4OHT-treated 39M1-81-ΔRaf-1:ER cells is biologically active. 39M1-81-ΔRaf-1:ER cells were infected with a recombinant lentivirus encoding an Egr-1 responsive reporter gene. The infected cells were stimulated with 4OHT (100 nM) for 16 hours. Cell extracts were prepared and analyzed for luciferase activities which were normalized to the protein concentrations.