Fig. 7. Expression of ERK2-MEK1-LA phosphorylates Elk-1 efficiently in suspended cells. NIH 3T3 cells were transfected with either pCMV5 (Vec), 22W Raf, or pCMV5-ERK2-MEK1-LA alone (A) or in combination with pCMV5-FLAG-Elk-1 (B). Serum-starved cells were either replated on fibronection (Fn) or maintained in suspension (Sus) for 2 hours in DMEM/BSA. (A) ERK activity was determined by immunoprecipitation with anti-myc antibody followed by western blotting with anti-active ERK antibodies and by in vitro kinase assay using recombinant GST-Elk-1 as the substrate. (B) Elk-1 phosphorylation was determined by immunoprecipition from cell lysates with an anti-FLAG antibody and western blotting for levels of Ser383 phosphorylated and total Elk-1. The results shown are representative of three separate experiments.