Fig. 4. Effects of B-Raf, B-RafAA or B-RafED on ERK activity and Elk-dependent transcription. (A) Activation of ERK by B-Raf, B-RafAA or B-RafED. Myc-ERK was co-transfected with pcDNA3, HA-tagged B-Raf, B-RafAA or B-RafED. Co-transfection of Myc-ERK and HRasV12 serves as a positive control (lane 2). Cells were cultured for 24 h followed by starvation for 15 h. Myc-ERK was immuno precipitated with anti-Myc monoclonal antibody 9E10 and assayed for kinase activity using GST–Elk1 as a substrate. ERK kinase activity is shown in the top panel and fold activation was determined by PhosphorImager analysis. Immunoblot detection of Myc-ERK or HA-B-Raf is shown in the middle or bottom panel. (B) Elk1-dependent transcription activation by B-Raf, B-RafAA or B-RafED. COS cells were transfected with Gal4-Elk1 and Gal4-LUC and B-Raf, B-RafAA or B-RafED. A pCMV-LacZ plasmid was co-transfected as an internal control for variations in transfection efficiency. Cells were cultured for 24 h followed by 15 h starvation. Luciferase activity was measured and normalized against the co-transfected β-galactosidase activity and results are mean ± SD from three independent experiments.