TLR ligands induce intracellular Ca2+ release and CaMKII activation in macrophages. (A) RAW264.7 cells were loaded with Fluo 3/AM and stimulated with 0.1 μg/mL LPS in the absence or presence of 1.5 mM ethyleneglycoltetraacetic acid or 6 μM BAPTA-AM, then imaged by Leica TCS SP2 confocal microscopy under a 10×/0.40 CS objective lens at 10-second intervals. The basal (left panel) and peak (middle panel) fluorescence is displayed. Graphs (right panel) showed changes in mean fluorescence intensity from 15 cells per microscopic field over time. Data are representative of 3 independent experiments (original magnification ×100). L indicates LPS. (B) Cell extracts of TLR ligand–stimulated RAW264.7 cells were immunoprecipitated with anti–CaMKII-α antibody. The immunoprecipitates were subjected to CaMKII activity assay with autocamtide-2 as substrate or immunoblotted with anti–CaMKII-α antibody. Data are shown as mean plus or minus SD of 3 independent experiments. *P < .05, **P < .01 vs unstimulated cells.
