Fig. 7. Inhibition of JNK and p38 kinase signaling by Rac2^D57N. (A) COS7 cells were co-transfected with HA-JNK2 and the indicated expression constructs. Cells were stimulated with EGF for 20 minutes or exposed to UV radiation. Data shown are a representative experiment of two independent experiments. (Top) Autoradiogram of an in vitro kinase assay by immunoprecipitated HA-JNK2 using GST-Jun as the substrate. A phosphoimager was used to measure levels of phosphorylated GST-Jun. Fold increases in phosphorylation of GST-Jun are relative to basal GST-Jun phosphorylation of COS7 cells co-transfected with HA-JNK2 and empty vector. (Middle, bottom) Immunoblots using an antibody to HA to detect the presence of HA-JNK2 and an antibody specific to Rac2 to measure Rac2 expression. (B) COS7 cells were co-transfected with FLAG-p38 kinase and the indicated expression constructs. Cells were stimulated with sorbitol for 30 minutes, or exposed to UV irradiation. Data shown are a representative blot from four independent experiments. (Top) A blot with an antibody to the phosphorylated form of p38 kinase. Fold increases in phosphorylation of p38 kinase are relative to basal phosphorylation of p38 kinase in COS7 cells co-transfected with FLAG-p38 kinase and empty vector. (Middle, bottom) Immunoblots using the FLAG antibody to detect FLAG-p38 kinase and an antibody specific to Rac2 to measure Rac2 expression.