Fig. 4. DAP kinase and MLCK display distinct roles in the assembly of stress fibers. (A) DAP kinase is insensitive to ML-7. ML-7 was added at indicated concentrations to kinase reactions containing MLC, Flag-DAP kinase and Ca²⁺/calmodulin. MLC phosphorylation (bottom) and DAP kinase autophosphorylation (top) were detected by autoradiography. (B) Kinase reactions as in (A) but without ML-7 were performed for various times. The extent of phosphorylation was quantified and expressed as percentage, assigning the maximum phosphorylation of MLC to 100%. (C) Virus-infected NIH3T3 cells (Fig. 2B) were serum starved and treated with 5 µM of ML-7 for 30 minutes. Cells were then fixed and stained with rhodaminephalloidin. (D) Subcellular localization of endogenous DAP kinase. NIH3T3 cells cultured in serum-containing medium were double stained with anti-DAP-kinase for endogenous DAP kinase (a) and with rhodamine-phalloidin for F-actin (b). The merged image is shown in (c). Scale bars, 10 µm.