Fig. 1

Shp1 localizes at invadopodia in A375MM cells. A Representative confocal images of A375MM cells showing colocalization of Shp1 with invadopodium markers. Cells were plated on fluorescent gelatin-coated coverslips in complete media, fixed and immunostained for cortactin and actin. Shp1, cortactin, actin and gelatin are shown in green, magenta, cyan and gray, respectively. Degraded ECM is shown as dark areas. The invadopodia are shown as cortactin- and actin-positive dots corresponding to the dark areas. Arrowheads indicate invadopodia colocalizing with Shp1. Confocal images are representative of at least four different experiments. Scale bars, 10 μm. B Shp1, Fak, cortactin and MT1-MMP protein expression were assayed by western blotting in cytosolic, plasma membrane and invadopodia-enriched subcellular fractions extracted from lysates of A375MM cells incubated for 24 h on a thick layer of cross-linked gelatin, as described in “Methods” section. The invadopodia markers cortactin and MT1-MMP were enriched in the invadopodia fraction. Protein loading was assessed by Ponceau red staining. Molecular weight standards (kDa) are indicated on the left of each panel. Representative western blot images of two independent experiments