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Fig. 3. Inhibition of Arp 2/3 or cofilin function causes a reduction in barbed ends. Cells were time-lapsed after addition of EGF and the fluorescent edge intensity (GFP-ßactin) was measured every 10 seconds and barbed ends calculated (see Materials and Methods). (A) Fold GFP-actin edge intensity increase after addition of EGF for nonimmune IgG-injected cells (n=15); (B) for anti-p34-injected cells (n=18); (C) for anti-cofilin-injected cells (n=17) (error bars: ±s.e.m.). (D) The relative increase in barbed ends at the leading edge after stimulation. Barbed ends were calculated by determining the slopes of the initial fluorescence intensity increase (10-100 seconds after EGF stimulation) as a measure of the rate of GFP-ßactin incorporation (Lorenz et al., 2004). The first bar shows barbed ends present after injection of anti-p34, i.e. essentially the cofilin contribution to barbed ends. The second bar indicates barbed ends present after injection of anti-cofilin, i.e. the Arp2/3 contribution to barbed ends. The last bar shows barbed ends present in control IgG-injected cells (full length of the bar). The overlay (dark area on the bar) corresponds to the calculated sum of cofilin and Arp2/3 contributions as displayed in the first two bars. To estimate error bars for D, slopes were determined for straight lines that were calculated using the data in A-C±s.e.m.
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