Cdc42 and RhoA regulate the shear stress activation of JNK. (a) HA-JNK1 (1 μg/slide) was cotransfected with pcDNA3, HA-Cdc42(N17), or HA-RhoA(N19) (2 μg/slide) into BAECs cultured on glass slides. The total amount of the transfected plasmid DNA in the various samples was kept constant by supplementing with pcDNA3. About 40 h after transfection, the confluent BAEC monolayers were either sheared at 12 dyn/cm² for 30 min or kept under static conditions for 30 min. The cell lysates were immunoprecipitated with a polyclonal anti-JNK1 antibody, and GST-c-Jun(1-79) and (γ-³²P)ATP were added to the immunoprecipitates for immunocomplex kinase assays. The bands indicated by the arrow represent the phosphorylated GST-c-Jun(1-79). Shown at bottom is the immunoblotting with anti-HA mAb indicating that the levels of HA-JNK1 were comparable among the various samples. The expression levels of transfected HA-Cdc42(N17) and HA-RhoA(N19) were also shown in the immunoblot. (b) Confluent monolayers of BAECs were treated with or without 6.5 μg/ml recombinant C3 for 48 h. The cells were then either sheared at 12 dyn/cm² for 30 min or kept under static conditions, followed by JNK1 kinase activity assays. The bar graphs, representing mean ± SD from three separate experiments, show the JNK kinase activity of the various samples relative to that in the pcDNA-transfected cells and static controls in a, and relative to that in the static control without C3 pretreatment in b. Asterisks in a indicate significant difference (P < 0.05) compared with pcDNA3-transfected cells after shearing. Asterisks in b indicate significant difference (P < 0.05) compared with control cells after shearing.