Fig. 2. DAP kinase promotes the assembly or stabilization of stress fibers. (A) NIH3T3 cells were infected with recombinant retrovirus carrying vector alone or various forms of DAP kinase as indicated. Infected cells were selected by puromycin and then subjected to immunoblot analysis to detect the expression of DAP kinase proteins. (B) Cells as in (A) were serum-starved for 6 hours, treated with or without 25 mM BDM for 10 minutes or 100 nM cytochalasin D for 30 minutes, and then stained with rhodamine-phalloidin. (C) Quantitation of cells with stress fibers in experiments as described in (B). Only cells that were not exposed to BDM and cytochalasin D were analysed. The values shown are means ± s.d. from at least three independent experiments and more than 300 individual cells were counted for each experiment. (D) NIH3T3 cells transiently transfected with DAP kinase were serum starved and then double stained with anti-DAP-kinase and rhodamine-phalloidin. A cell that overexpresses DAP kinase is indicated by strong DAP kinase staining pattern. (E) NIH3T3 cells were cotransfected with DAP kinase, MLCS19A mutant (or a control vector) and GFP at a ratio of 5:5:1. Cells were serum starved and stained with rhodamine-phalloidin. (F) Quantitation of GFP-positive cells with stress fibers in experiments described in (E). Scale bars, 10 µm.