Figure 5.

ET-1 induces JNKs and p38MAPK phosphorylation in rat cortical astrocytes. A, B, Time course of ET-1-induced JNK (A) and p38MAPK (B) phosphorylation. Cells were stimulated with 100 nm ET-1 for the indicated times, and total cell lysates were analyzed by Western blot (as described in Materials and Methods) using an anti-P-JNK antibody (A, top) or an anti-P-p38MAPK antibody (B, top). The same blots were then stripped and incubated with phosphorylation-state independent anti-JNK or anti-p38MAPK antibodies to normalize for the total (phosphorylated plus nonphosphorylated) amounts of JNKs (A, bottom) and p38MAPK (B, bottom). C, ET-1 induces JNK phosphorylation via the upstream kinase MLK. Cells were preincubated for 30 min with the MLK inhibitor CEP-11004 (5 mm) and then treated with 100 nm ET-1 for 20 min. Total cell lysates were then analyzed for P-JNK phosphorylation (C, top) or total JNK (C, bottom) as described above. Data are representative of three independent experiments.