Fig. 2. Activated JNK phosphorylates the nuclear transcription factor c-Jun regardless of adhesion. Serum-starved detached NIH 3T3 cells were either plated onto fibronectin (Fn)-coated coverslips or maintained in suspension (Sus) for 2 hours in DMEM/BSA. At this time, cells were stimulated appropriately with anisomycin (50 ng/ml) for 30 minutes. Suspended cells were plated on poly-lysine-coated coverslips for the final 15 minutes. Cells were washed briefly in PBS before fixation. Cells were processed by immunofluorescence for phospho-JNK (A), phosphoserine-63 c-Jun (B), and total c-Jun (B,C). Primary antibody staining was detected with the appropriate fluorophore-conjugated anti-rabbit and anti-mouse IgG. In A, inserts show the overlay between phospho-JNK and nuclear staining with Hoechst 33342 reagent. Bars, 20 µM (A); 50 µM (B,C).