WAVE2 siRNA inhibits lamellipod protrusion after EGF stimulation. GFP–β actin–expressing MTLn3 cells were treated with no oligofectamine (control), scrambled siRNA, rat-specific WAVE2 siRNA, or rat-specific WAVE2 siRNA + human WAVE2 plasmid (W2 rescue). (A) Representative time-lapse images of cells at 0 and 3 min after EGF stimulation. Bar, 10 μm. (B) Representative images of filopods being scored are indicated by arrows in scrambled control cells (left) and WAVE2 KD cells (right). (C) Western blot analysis of cells treated with WAVE2 rat-specific siRNA (W2si) or Scrambled siRNA (Scr) in cells with control plasmid (C) or WAVE2 human plasmid (W2 wt). Westerns were blotted with α-WAVE2 and α–β actin antibodies. (D) Quantification of cell area fold increase during lamellipod extension. Graph shows the fold increase in area of control, scrambled siRNA, WAVE2 siRNA, and human WAVE2-expressing cells treated with WAVE2 siRNA. Results are from a total of 67 cells from five independent experiments. (E) Quantification of filopod formation after EGF stimulation. Results are from a total of 43 cells from six independent experiments. (F) Knocking down Abi1 results in the destabilization of the WAVE2 complex and a WAVE2 KD phenotype. Representative Western blot of Abi1 KD at 48 h. Westerns were blotted with α-WAVE2, anti-Abi1 α-tubulin, and α–β actin antibodies. (G) Quantification of lamellipod extension at 0, 1, and 3 min. (H) Quantification of filopod formation. Error bars indicate ±SEM of 27 cells from three independent experiments.