Figure 3

Over-expression of wild type or dominant negative LIMK1 regulates cofilin phosphorylation and actin polymerization in Ra2 cells. Ra2 cells received increasing doses (80, 150, or 200 equivalents) of lentivector expressing LIMK1-WT or LIMK1-DN. A) Immunofluorescence of doxycycline-induced Ra2 cell populations using Alexa488-conjugated phalloidin (green) and anti-cofilin-ser3(P) rabbit antibodies (red). Detection gain was adjusted to include the signal for phalloidin or cofilin-ser3(P) from all Ra2 cell populations within the dynamic range and was then kept constant throughout. Bars, 10 μm. B) Expression of LIMK1 protein and effect on phosphorylation of cofilin was determined by western blotting with polyclonal rabbit anti-LIMK1 antibodies and anti-Ser3(P)-cofilin antibodies. Ra2 045 control cells express only the tetracycline-responsive rtTA-transactivator protein. Note that the LIMK1 antibody used only recognizes transgene human LIMK1 protein. C and D) Mean and SEM integrated optical density of western blot bands of LIMK protein (C) and Ser3(P)-cofilin (D) of three individual experiments performed as in B. E and F) The F/G-actin ratios of LIMK1-expressing cells (E) or toxin-treated control cells (F) represent mean and SEM of at least three independent experiments.