FIG. 4.

JNK activation is involved in TNF-α killing of MCF-7 cells. (A) MCF-7 cells were pretreated with SP600125 (20 μM) for 30 min and stimulated with TNF-α (20 ng/ml) for 15 min. JNK was immunoprecipitated with anti-JNK1 antibody, and its activity was measured by immune complex kinase assay with GST-c-Jun(1-79) as the substrate. JNK content was analyzed by immunoblotting with anti-JNK antibody. The same cell lysates were also analyzed for activation of p38 or ERK by immunoblotting with corresponding anti-phospho antibodies. (B) MCF-7 cells were pretreated with SP600125 (SP; 20 μM) for 30 min and stimulated with TNF-α (20 ng/ml) for 11 h. Apoptotic cell death was calculated as described in the legend to Fig. 2C. (C) MCF-7 cells were cotransfected with expression vectors encoding GFP (0.5 μg) and either the HA-JNKK2-JNK1 fusion protein, the dominant negative mutant HA-JNKK2(K149M), or an empty vector (2.0 μg of each). Cells were treated with or without TNF-α (20 ng/ml) for 11 h and stained with Hoechst. The death of transfected (GFP-positive) cells was calculated as described in the legend to Fig. 1.