Overexpression of constitutively active CaMKII enhances TLR-triggered proinflammatory cytokine and IFN-β production in macrophages. RAW264.7 cells (1.5 × 105) (A,C,D) were transiently transfected with constitutively active CaMKII plasmid (CaMKII290). Mouse peritoneal macrophages (4 × 105) (B) were nucleofected with CaMKII290 using Amaxa Nucleofector II Biosystems. After 36 hours, the cells were stimulated with 0.1 μg/mL LPS (A,B), 0.3 μM CpG ODN (C), or 10 μg/mL poly(I:C) (D), respectively, for the indicated time. IL-6, TNF-α, or IFN-β in the supernatants was detected by ELISA. Data are shown as mean plus or minus SD of 3 independent experiments. HEK293 cells were cotransfected with 50 ng of MyD88 (E) or TRIF (F,G) expressing plasmid, 50 ng of TNF-α (E,G), or IFN-β (F) luciferase reporter plasmid, 10 ng of pTK-Renilla luciferase, together with indicated amount of CaMKII290 expressing plasmid. Total amounts of plasmid DNA were equalized using empty control vector. After 24 hours of culture, luciferase activity was measured and normalized by Renilla luciferase activity. The expression of CaMKII290 in HEK293 cells was immunoblotted with anti-flag antibody (E). Data are shown as mean plus or minus SD (n = 5) of one typical experiment from 3 independent experiments with similar results. *P < .05; **P < .01.
