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).
