FIG. 4.

Expression and activation of c-Jun are impaired in Jnk1 null fibroblasts. (A) The levels of c-Jun expressed were measured by immunoblotting (IB) using anti-c-Jun antibody (α-c-Jun). (B) Cells were treated with TNF-α (5 ng/ml) for 30 min (+) or not treated with TNF-α (−). c-Jun phosphorylation at Ser63 and Ser73, as well as the levels of protein expressed, were measured by immunoblotting using anti-phospho-c-Jun(Ser63), anti-phospho-c-Jun(Ser73), and anti-c-Jun antibodies. (C) As in panel B, except that the cells were treated with TNF-α for 1 h. (D) Cells were treated with UV (60 J/m²) for 1 h (+) or not treated with UV (−). c-Jun expression and phosphorylation were measured as described for panel B. (E) Cells were transfected with expression vectors encoding GAL4-Luc (0.5 μg), along with GAL4-c-Jun or empty vector (10 ng each), and treated with TNF-α (5 ng/ml) for 10 h (+) or not treated with TNF-α (−). GAL4-Luc activity was measured as described previously (31). (F) Activation and levels of c-Jun expressed in early passage WT, Jnk1^−/−, and Jnk2^−/− primary MEFs (passage 3) treated by TNF-α as described for panel B. (G) The levels of c-Jun protein expressed and activation of c-Jun by treatment with TNF-α (5 ng/ml) for 30 min in late passage WT, Jnk1^−/−, and Jnk2^−/− primary MEFs (passage 8) were analyzed as described for panel B.