Fig. 4. Phosphorylation of Elk-1 is anchorage-dependent upon activation of the ERK pathway, but anchorage-independent upon activation of the JNK pathway. (A) NIH 3T3 cells were transfected with either MEKK1 or 22W Raf. Cells were replated onto fibronectin-coated coverslips. The localization of MEKK1 and Raf were determined by immunofluorescence using either TRITC or FITC-conjugated anti-rabbit secondary antibodies. Bar, 25 µM. (B) Cells expressing FLAG-Elk-1 were plated onto Fn-coated dishes or maintained in suspension (Sus) and analyzed by immunofluorescence with anti-Elk-1 antibodies. Insets show the overlay of Elk-1 and nuclear staining. (C) Cells were transfected with FLAG-Elk-1 and either vector 22W Raf or MEKK1. After 24 hours, transfected cells were serum-starved before being replated in DMEM/BSA onto Fn-coated dishes or maintained in suspension (Sus) for a further 3 hours. Ectopically expressed Elk-1 was immunoprecipitated from cell lysates from each condition with an M2 FLAG epitope antibody. FLAG-Elk-1 immunoprecipitates were analyzed by western blotting for levels of Ser383-phosphorylated and total Elk-1. The results shown are representative of at least three independent experiments with equivalent results.