Role of PKCζ in eNOS protein stability. (A) Schematic diagram showing experimental protocol. (B-C) Ad.DN-PKCζ and Ad.ERK5-S486A increase eNOS protein stability. HUVECs were infected with Ad.LacZ (control) or Ad.DN-PKCζ (B) and Ad.ERK5-WT or Ad.ERK5-S486A (C). Sixteen hours later, the cells were incubated with TNFα 10 ng/mL plus the protein synthesis inhibitor cycloheximide (CHX; 10 μg/mL). HUVECs were harvested after 0, 4, 8 and 12 hours of treatment, and Western blots were performed with eNOS, PKCζ, ERK5, and tubulin antibodies. (D-E) Western blots were quantified by densitometry by setting the time zero to 1.0. Data presented are from a representative experiment of at least 3 independent experiments. *P < .1; **P < .05.