Figure 5

Cofilin knock-down increases F/G-actin ratio and inhibits superoxide production. A and B) Levels of cofilin protein after tetracycline induction of two different cofilin shRNAs alone or in combination was determined by western blotting. Control cells refer to Ra2 cells transduced with empty pLVTHM dsRed vector. The bar graphs in B shows mean optical density and SEM of western blot bands derived from three independent experiments. C) Ratio of F- to G-actin in control and cofilin-1 shRNA-expressing Ra2 cells. Data represent mean ± SEM of two independent experiments, each performed in triplicate. D-G) Superoxide production in control or cofilin shRNA-transduced Ra2 cells before and after stimulation with FMLP (D,E) or PMA (F,G) was measured by luminol E-CL. The ordinate of the trace graphs show luminol E-CL normalized to control cells in arbitrary units. Injection of agonist is indicated by arrow. Bar graphs E and G) show normalized luminol E-CL mean and SEM of three independent experiments as performed in A or C, respectively.