Fig. 4. The Rac mitogenic rescue implicates both a JNK and a Nox activity. (A) Structure-function analysis of the Rac mitogenic rescue. (B) The Rac mitogenic rescue is sensitive to inhibitors of JNK and Nox activities. (C) The biological defect of RacL61/N52L is compensated by activators of the JNK pathway. (D) The biological defect of the RacL61/D38N is compensated by an activator of the Nox pathway. Quiescent NIH3T3 transfected with Abl-PP-K^– together or not with indicated constructs were stimulated or not with mitogen (20 ng/ml PDGF or 10% serum as shown) in the presence of BrdU and proceeded as in Fig. 2. In panel B, cells were treated with 0.5 µM SP600125, 20 µM SB203580, 0.5 µM DPI or 10,000 U catalase as indicated before stimulation. The mean±s.d. of the percentage of BrdU-positive cells present in expressing and non-expressing cells under the specified conditions is shown. (E) Activity of various RacL61 mutants used in this study. NIH3T3 infected with control or indicated RacL61 retroviruses were incubated overnight with 0.5% serum and assayed for Rac activity as described in Fig. 1. Total Rac level and Rac activity (Rac-GTP) is shown. (F) In vivo activity of DA-MKK4 and 7 used in this study. Cells were transiently transfected with indicated DA-MKK and Flag-tagged JNK1 constructs as indicated. Immunoprecipitated Flag-JNK was assayed for in vitro activity as described in Materials and Methods. The level of phosphorylated Jun (³²P-Gst-Jun) is shown.