RAW264.7 cells stably expressing empty vector, FLAG-tagged PKD1, FLAG-tagged PKD2, or FLAG-tagged PKD3 were stimulated with medium, CpG-B DNA (12 μg/ml) for 45 min. Each PKD family protein in whole cell lysates was immunoprecipitated with anti-FLAG Ab. Kinase activity of PKDs was analyzed by in vitro kinase assay using syntide-2 as a PKD substrate (top). Expression and phosphorylation status of each PKD was analyzed by immunoblotting with anti-FLAG and anti-phospho-PKD Abs, respectively (bottom).