Fig. 1. Anisomycin treatment activates JNK regardless of the state of anchorage. Serum-starved detached NIH 3T3 cells were either maintained in suspension (Sus) or plated onto fibronectin (Fn)-coated dishes for 2 hours in DMEM/BSA. (A) Cells at the 2 hour time point were stimulated appropriately with increasing doses of anisomycin (0, 5, 20, 50 and 100 ng/ml) for either 15 minutes or 30 minutes, as indicated. (B) Cells were stimulated with 50 ng/ml anisomycin for 0, 5, 15, 30, 45 and 60 minutes, as indicated. Under each condition, cells were lysed in modified RIPA buffer and endogenous JNK activity determined by immunoprecipitation with C-17 and in vitro kinase assay using GST-c-Jun as the substrate. Incorporation of ³²P into GST-c-Jun was determined by autoradiography.