Fig. 5.

Met-RTK-driven invadopodia biogenesis is dependent on two PxxP motifs in Gab1. (A) Schematic diagram of Gab1, indicating Met binding domain (MBD) and a proline-rich region 4/5 (P4/5) and sites of recruitment for downstream signaling proteins. PH; pleckstrin homology domain. (B) Gab1 null Tpr-Met cells rescued with either WT Gab1, Gab1ΔMBD or Gab1ΔP4/5 were cultured on gelatin matrix for 24 hours, fixed, stained with phalloidin and confocal images acquired. Arrows indicate rosettes and arrowheads indicate matrix remodeling. (C) Cells rescued with either WT Gab1 or the indicated Gab1 mutants were subjected to Boyden chamber invasion assays. Representative images are shown. (D,E) Quantification of invadopodia response (D) and Boyden chamber invasion response (E) is shown for three clones for each mutant and WT Gab1-rescued cells. Values are the means of three independent experiments. (F) SDS-PAGE was performed on lysates from the corresponding rescue cells and probed for GFP, Met-P (pMet), Met and tubulin. Scale bars: 10 μm.