Fig. 7. DAP kinase does not stimulate the formation of focal adhesions. (A,B) NIH3T3 cells co-transfected with various DAP kinase constructs and GFP at a ratio of 10:1 were serum starved for 6 hours and then stained with anti-paxillin (A) or anti-vinculin (B). The transfected cells are visualized by GFP fluorescence. (C) Quantitation of cells with focal adhesions in experiments as described in (A). 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 infected with recombinant retrovirus carrying DAP kinase or DAPKCaM were serum starved and then double stained with rhodamine-phalloidin (red) and anti-paxillin or anti-vinculin (green). Alternatively, NIH3T3 cells were treated with 200 ng ml^–1 LPA for 15 minutes and then subjected to the same immunostaining. Images shown are the superimpositions of red and green fluorescence. (E) NIH3T3 cells transfected with DAP kinase were serum starved for 6 hours and then triple stained with rhodamine-phalloidin (a), anti-paxillin followed by FITC-conjugated anti-mouse secondary antibody (b) and anti-DAP-kinase followed by AMCA-conjugated anti-rabbit secondary antibody (c). The overlay of three images is shown in (d). Scale bars, 10 µm.

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