TNFα inhibits ERK5 transactivation by PKCζ. (A) HA-PKCζ-WT and CATζ overexpression was verified with the use of cell lysates probed with anti-PKCζ. (B) PKCζ-WT and CATζ inhibit ERK5 transactivation. HUVECs were transfected with pBIND-ERK5 and Gal4-dependent (pG5-Luc) reporter gene with or without pcDNA3-CA-MEK5α with 0.3 μg of control vector or expression plasmids for PKCζ-WT or CATζ. ERK5 transcriptional activity was evaluated by measuring luciferase activity after 24 hours. (C) PKCζ silencing was confirmed by Western blot analysis. (D) Depletion of PKCζ expression reverses TNFα-mediated inhibition of ERK5 transcriptional activity. HUVECs were pretreated for 24 hours with siRNA targeting PKCζ and then cotransfected with pBIND-ERK5 and Gal4-dependent (pG5-Luc) reporter gene with or without pcDNA3-CA-MEK5α. After 8 hours, cells were treated with 10 ng/mL TNFα, and ERK5 luciferase activity was determined 16 hours later. Results are expressed in arbitrary units normalized to the control (set to 1.0 for each experiment). Data are mean ± SD of 3 experiments performed in triplicate. **P < .05.