The ERK5-CHIP signal module protects endothelial apoptosis in response to 15d-PGJ₂. (A) HUVECs were pretreated with DMSO or 10 µM BIX02189, a specific inhibitor of MEK5, before applying L-flow. Cells were then exposed to 10 µM 15d-PGJ₂ for 8 h. The protein expression of p53, phosphorylated ERK5, and tubulin were determined by immunoblotting with specific antibodies. (B) HUVECs were transfected with siRNA against human CHIP (si-CHIP) or control RNA and then followed by transducing the adenovirus encoding con stitutively active form of MEK5 alpha (Ad-CA-MEK5α). Cells were then exposed to 10 µM 15d-PGJ₂ for 8 h. Cell lysates were subjected to Western blot analysis with anti-cleaved caspase 3, anti-phospho ERK5, anti-CHIP, and anti-tubulin antibodies. (C) Cell viability was determined by MTT assay. Data are expressed as the mean±SD from three independent experiments. ^*P<0.01. ERK5, extracellular signal-regulated kinase 5; CHIP, C terminus of Hsc70-interacting protein; 15d-PGJ₂, 15-deoxy-Δ(12,14)-prostaglandin J₂; HUVEC, human umbilical vein endothelial cells; OD, optical density; NS, non-significant.