CaMKII directly binds, phosphorylates IRF3, and enhances IRF3-activated IFN-β expression. (A,B) RAW264.7 cells were stimulated with 0.1 μg/mL LPS for the indicated time. Equal amount cell lysates were immunoprecipitated with CaMKII-α (A) or IRF3 (B) antibody and then detected with CaMKII-α and IRF3 antibody. (C) HEK293 cells were transfected with IRF3-HA and wt or mutant CaMKII-α–flag construct as described in Figure 5C. After 24 hours, cell extracts were immunoprecipitated with anti-flag antibody and then detected with anti-HA and anti-flag antibody. (D) CaMKII-α–flag construct together with HA-tagged IRF3, IRF3N140, or IRF3C141 plasmid were transfected into HEK293 cells. After 24 hours, IRF3 truncates were immunoprecipitated with HA-specific antibody. Precipitated proteins were detected by immunoblot. (E) GST pull-down assays were performed with GST-tagged CaMKII-α and RAW264.7 cell lysates. (F) wt or mutant GST-IRF3 380-427 were used as substrates of recombinant active CaMKII-α. The incorporation of ³²P in the IRF3 380-427 was visualized by autoradiography after SDS-PAGE. Residues are as follows: 2A, S385A, S386A; 5A, S396A, S398A, S402A, T404A, S405A; 7A, S385A, S386A, S396A, S398A, S402A, T404A, S405A. Data are representative of 3 independent experiments. (G) HEK293 cells were transfected with 100 ng of IRF3-expressing plasmid, 50 ng of IFN-β luciferase reporter plasmid, 10 ng of pTK-Renilla luciferase, together with indicated amount of CaMKII290 plasmid. After 24 hours, luciferase activity was measured and normalized by Renilla luciferase activity. Data are shown as mean plus or minus SD (n = 5) of 1 typical experiment from 3 independent experiments with similar results. *P < .05; **P < .01.