Flow and TNF modify phosphorylation of ASK1 Ser-967. (a) A model for regulation of ASK1 activity by TNF and flow. ASK1 Ser-967 is in a phosphorylated state (pSer-967). TNF induces dephosphorylation of pSer-967 to release ASK1 from inhibitor 14-3-3. ASK1 dimerization results in activation of the ASK1-JNK cascade. Flow prevents TNF-induced dephosphorylation of pSer-967 to block release of ASK1 from 14-3-3. (b) GST–14-3-3 binding assay for phosphorylation of ASK1 Ser-967. HA-tagged ASK1-WT was transfected into ECs, and pSer-967 of ASK1 was determined by an in vitro binding to GST–14-3-3 followed by Western blot analysis using anti-HA. GST, GST–14-3-3-K49A (a phosphoserine binding–deficient mutant) were used as controls. (c) Flow and TNF regulate phosphorylation of ASK1 Ser-967. ECs were transfected with ASK-WT. Twenty-four hours after transfection, cells were subjected to “preconditioning” protocol. Amount of phosphorylated ASK1 Ser-967 was measured by an in vitro GST–14-3-3 binding assay. Data are representative of two experiments in HUVECs.