Flow pre-exposure inhibits TNF-mediated JNK and ASK1, but not NF-κB, activation in ECs. ECs were subjected to the following “preconditioning” protocol: they were maintained in static conditions for 25 minutes (Ctrl), exposed to flow for 10 minutes and then held static for 15 minutes (Flow), maintained in static conditions for 10 minutes, followed by TNF stimulation for 15 minutes or subjected to flow for 10 minutes followed by TNF-α (100 U/ml) stimulation for 15 minutes (flow + TNF). (a) Flow inhibits JNK activation by TNF. Cell lysates were prepared and analyzed for JNK activity by an in vitro kinase assay using GST–c-Jun as a substrate. (b) Flow has no inhibitory effect on NF-κB. Cells were treated as in a. Nuclear extracts from these cells were used for EMSA with a κB probe. Specificity of NF-κB complex was verified by 50-fold molar excess of the unlabeled κB oligonucleotide in the TNF-treated sample (TNF + competitor). (c) Flow inhibits ASK1 activation by TNF. Cell lysates in a were prepared and analyzed for ASK1 activity by an in vitro kinase assay using GST-MKK4 as a substrate. Autoradiograms shown in a–c are representative of three experiments in HUVECs.