Time course of MAP kinase activation by ABL-N. MDA-MB-231 cells were treated with vehicle (0.5% ethanol) or 20 μM of ABL-N and harvested at the indicated times. (a) JNK; (b) p38; and (e) ERK activation was determined by Western blot analysis using antibodies that recognize the phosphorylated form of the respective active MAP kinase. (c) JNK activity was determined by an in vitro kinase assay as described in Materials and methods. Phosphorylation of c-Jun, which represents the intrinsic activity of JNK, was visualized by Western blotting. (d) ABL-N (20 μM) and GST-JNK1 fusion proteins (1 μg) were incubated for 12 hours or cells were treated with ABL-N (20 μM) for 12 hours and lysates were prepared, then JNK activity was measured as described above. Phosphorylation of c-Jun was visualized by Western blotting. The same blot was stripped and reprobed with a c-Jun antibody to monitor equal loading of proteins. Data are representative of three independent experiments.