Fig. 8. LIMK1 siRNA has no effect on EGF-stimulated lamellipod protrusion and barbed-end formation. (A) MTLn3 cells were treated with control or LIMK1-specific siRNA oligonucleotides for 36 hours prior to EGF stimulation. Cell lysates were prepared at 0 and 60 seconds following EGF stimulation, western blotted and probed with antibodies against p-cofilin and LIMK-1. (B) Quantification of western blots showing the decrease of LIMK1 expression and cofilin phosphorylation levels in control (left) and LIMK1 siRNA treated cells (right). Error bars indicate the standard error of the mean. (n=3 separate experiments). (C) The change in protrusion area following EGF stimulation of MTLn3 cells is not affected by LIMK1 siRNA treatment. Time lapse microscopy of MTLn3 cells demonstrate no change in the initial rate or final extent of lamellipod protrusion in LIMK1 siRNA-treated cells () compared to control cells () following stimulation with EGF. (D) The increase in free barbed ends following EGF stimulation of MTLn3 cells is unaffected in LIMK1 siRNA. Time lapse microscopy of ß-actin-GFP MTLn3 cells demonstrate there is no effect on the fold change in the appearance of new barbed ends in LIMK1 siRNA-treated cells following EGF stimulation compared to control cells. For panel C and D, the error bars indicate the s.e.m. (n=2 separate experiments).