FIG. 4.

Akt diminishes ASK1-mediated JNK and ATF-2 activation. (A) Akt activity decreased ASK1-dependent JNK activity. 293 cells were transiently transfected for 24 h with 1 μg each of the indicated constructs, serum starved for 6 h, and treated with 100 ng of IGF-1/ml for 15 min. GST-JNK3 was immunoprecipitated (IP) from cell lysates and immunoblotted (W) with anti-phospho-JNK (anti-pJNK). After immunoblots were stripped, GST-JNK3 protein was identified with anti-GST. Crude lysates were probed with anti-HA to determine ASK1 protein levels and with anti-Akt to determine Akt protein levels. Data are representative of three independent experiments. (B) Akt decreased ASK1-induced ATF-2 activity. 293 cells were transiently transfected with ATF-2 luciferase plasmids and the indicated constructs for 24 h and then incubated in 1% FBS for 24 h. Cells were lysed and assayed for luciferase activity. The fold activation was determined in reference to the vector (pcDNA3) alone (=1.0). All bars represent means plus SEM from three to five independent experiments. The asterisk indicates a significant difference from ASK1 at P < 0.01 by one-way ANOVA followed by the Bonferroni t test.