Activation of CaMKII enhances MAPK, NF-κB, and IRF3 activation in TLR-triggered macrophages. (A) RAW264.7 cells were pretreated with 15 μM KN62 (left panel) for 30 minutes or transfected with CaMKII290 plasmid and then cultured for 36 hours (right panel). The cells were stimulated with 0.1 μg/mL LPS for the indicated time. Phospho-ERK, JNK, p38, IκBα, and total ERK were detected by immunoblot. (B) RAW264.7 cells were transfected with 100 ng of pGL3.5XκB-luciferase, 10 ng of pTK–Renilla luciferase, together without (left panel) or with indicated amount of CaMKII290 plasmid (right panel). After 36 hours, the cells were stimulated with 0.1 μg/mL LPS for 6 hours. Luciferase activity was measured and normalized by Renilla luciferase activity. (C,D) RAW264.7 cells were treated as described in panel A; the whole cell extracts (C) or nuclear extracts (D) were prepared. Phospho- (C) or total-IRF3 (D) was detected by immunoblot. Data are representative of 3 separate experiments. (E) RAW264.7 cells were transfected with 100 ng of IRF3 luciferase reporter plasmids (80 ng of Gal4 luciferase reporter plasmid, 20 ng of Gal4-IRF3–expressing plasmid), 10 ng of pTK-Renilla luciferase, together with indicated amount of CaMKII290 plasmid. After 36 hours, the cells were stimulated with 0.1 μg/mL LPS for 6 hours. Luciferase activity was measured. Data are shown as mean plus or minus SD (n = 5) of one typical result from 3 independent experiments with similar results. *P < .05; **P < .01.