Fig. 6. DAP kinase is required for serum-induced stress fiber formation and is activated by serum. (A) Reduction of endogenous DAP kinase expression by siRNA. Cell lysates from untreated (–), control-siRNA- or DAPK-siRNA-treated cells were immnuoblotted with antibodies as indicated. (B) siRNA-mediated knockdown of DAP kinase inhibits stress-fiber formation in response to serum. Cells as in (A) were stimulated with serum for 20 minutes and then stained with rhodamine-phalloidin. Cells receiving siRNA were visualized by their FITC fluorescence. Scale bar, 10 µm. (C) NIH3T3 cells were transfected with Flag-DAP kinase and stimulated with or without serum for various times. The Flag-DAP kinase was immunoprecipitated from cell lysates and subjected to in vitro kinase reactions using MLC as a substrate. The kinase reactions were resolved by SDS-PAGE, and MLC phosphorylation was detected by autoradiography (top). The same kinase reactions were subjected to immunoblot analysis to detect the precipitated Flag-DAP kinase (bottom). The result shown is a representative experiment from three independent experiments. (D) Quantification of DAP kinase activities in response to serum stimulation. Experiments were performed as in (C), and the amounts of phosphorylated MLC were normalized by those of DAP kinase input in the kinase reactions. Data are expressed as fold inductions relative to cells that were not stimulated by serum. Each value represents means ± s.d. from three independent experiments.

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