Ly49Q is involved in the regulation of MAP kinase activation after CpG stimulation. (A) Activation kinetics of p38 and JNK. RAW264 cells were treated with CpG (0.3 μM) for the indicated periods, and the total cell lysates were prepared by directly adding SDS sample buffer. The total lysates were separated by SDS–polyacrylamide gel electrophoresis, and the activation of p38 and JNK was examined by Western blotting. (B) The signal intensity (pixel numbers) of phosphorylated MAP kinases was normalized to that of each total MAP kinase, and semiquantitative values for the MAP kinase activation are shown. (C) Intracellular distribution of phosphorylated p38 and JNK in the presence of CpG stimulation. FLAG-tagged Ly49Q expression plasmids were introduced into Ly49Q^hi RAW264 cells, and 24 hours after transfection, the cells were incubated with unlabeled CpG (0.3 μM) for 2 hours. The cells were then fixed and stained with antibodies against FLAG and phosphorylated p38. To analyze the distribution of phosphorylated JNK, Ly49Q^hi RAW264 cells were treated with CpG for 2 hours and stained with antibodies against phospho-JNK and LAMP-1.