Fig. 3. Phosphorylation of c-Jun by activated JNK is adhesion independent over a range of anisomycin concentrations. Serum-starved detached NIH 3T3 cells were either plated onto fibronectin (Fn)-coated dishes or maintained in suspension (Sus) for 2 hours in DMEM/BSA. At this time point, cells were stimulated (A) with increasing doses of anisomycin (0, 5, 20, 50 and 100 ng/ml) for 30 minutes or (B) with 50 ng/ml anisomycin for the times indicated. Under each condition, cells were lysed in modified RIPA buffer and lysates analyzed by western blotting for phosphoserine-63 c-Jun (Phospho63-cJun), phosphoserine-73 c-Jun (Phospho 73-cJun) and total c-Jun. The results shown are representative of three independent experiments.