FIG. 7.
c-N-Ras downregulates the activation of JNK through a distinct set of downstream targets. (A) N-Ras knockout and control N+/+ cells, c-N-Ras reconstituted cells [N−/−(2)/wtN43A], and N-Ras knockout cells stably expressing the switch 1 mutant N-Ras constructs were left untreated or treated with 1 ng of TNF-α per ml in the presence of 2 μg of cycloheximide (CHX) per ml. At the indicated times, cell lysates were prepared and 50 μg of each lysate was electrophoresed on SDS-10% polyacrylamide gels and transferred to PVDF. The blot was developed with anti-phospho-JNK monoclonal antibody and anti-mouse-HRP secondary antibody as described in the legend to Fig. 2A. Bands were visualized by standard ECL techniques. The results are representative of three separate experiments. (B) Cells (as described for panel A) were left untreated or treated with TNF-α and cycloheximide as described for panel A. A 50-μg portion of each lysate was electrophoresed and transferred as described previously. The blot was developed with anti-phospho-c-Jun (Ser63) and anti-mouse secondary antibody-HRP. Detection was performed using standard ECL techniques. The results are representative of three separate experiments. (C) Cells were left untreated or treated with TNF-α and cycloheximide for 4 h as described for panel A. At 4 h of treatment, the cells were harvested and lysed for analysis of the level of apoptosis by a Cell Death Detection ELISA Plus as described in the legend to Fig. 1B and Materials and Methods. Assays were performed in triplicate, and the results are plotted with error bars representing the sample standard deviation of the mean (σn − 1). The results are representative of two separate experiments.