Adhesion to fibronectin and EGF collaborate to provide efficient phosphorylation of the Elk-1 transcription factor. NIH 3T3 cells were transfected with either pCMV5 (Vector) or pCMV5-FLAG-Elk-1. In A, 1 μg of green fluorescent protein (GFP) was included in the transfections to identify transfected cells. After 48 h, transfected cells were serum starved before being replated in DMEM/BSA on fibronectin-coated coverslips (A) or maintained in suspension (Sus) or replated on fibronectin (Fn)-coated dishes for a further 3 h (B). (A) Localization of Elk-1 was determined by immunofluorescence with an Elk-1 antibody and TRITC-conjugated anti–rabbit secondary antibody. The scale bar depicts a 10 micron distance. (B) After the 3-h incubation, cells were treated with 20 ng/ml EGF for 15 min as indicated. Ectopically expressed Elk-1 was immunoprecipitated (IP) from cell lysates from each condition with an M2 FLAG epitope antibody. Immunoprecipitates were analyzed by Western blotting (WB) with antibodies to determine phosphorylated and total Elk-1 levels.
{}