Fig. 5. DAP kinase is involved in serum-induced stress-fiber formation. (A) NIH3T3 cells were infected with recombinant retrovirus carrying vector alone or DAPK42A, as indicated. Serum-starved cells were re-stimulated with serum for 20 minutes and then stained with rhodamine-phalloidin. Scale bar, 10 µm. The percentages of cells with stress fibers seen in serum-starved and stimulated conditions are indicated on the bottom. (B) NIH3T3 cells were transiently transfected with various combinations of constructs as indicated. Myc-ROCK and Myc-CAT were immunoprecipitated from cell lysates and then used to phosphorylate recombinant MLC in vitro in the presence or absence of 2 µM Y27632. 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 ROCK or CAT (middle). The expression of DAPK42A was detected by immunoblot (bottom). (C) Endogenous MLCK was immunoprecipitated from lysates of 293T cells transfected with various constructs, as indicated. The immunoprecipitates were used to phosphorylate recombinant MLC in vitro, in the presence or absence of 2 µM ML-7 (top). The same kinase reactions were subjected to immunoblot analysis to detect equal input of MLCK (middle). The expression of various DAP-kinase proteins was detected by immunoblot (bottom).