Figure 2.

Src activation at the heterotypic contact during TEM of melanoma cells. (A) Immunoblots of stable transfectants expressing GFP-tagged WT and DN Src in WM239 melanoma cells. The endogenous Src is indicated by an arrow and GFP-tagged Src by an arrowhead. (B) Confocal image showing the localization of WT-Src-GFP in melanoma cell transfectants. (C) Confocal images showing the concentration of WT-Src-GFP at the heterotypic contact during both cell attachment and transmigration. Arrows indicate the heterotypic contact between the transfected melanoma cell and endothelial cells. (D) Immunolocalization of activated Src (p-Src) at the heterotypic contact during TEM of nontransfected melanoma cell. (a and b) Cocultures fixed at 1 h showing melanoma cells at the attachment stage. (c and d) Cocultures fixed at 5 h showing the transmigration stage. Control cocultures (a and c) and cocultures pretreated for 5 min with 1 mM pervanadate before fixation (b and d) were immunostained for p-Src. Melanoma cells are marked by an asterisk. Arrows indicate the heterotypic contact between melanoma cells and endothelial cells and arrowheads indicate the homotypic endothelial cell junction. Bars, 10 μm (B, C, and D). (E) Immunoblots showing that PP2 abolished Src activation during TEM. Melanoma cells were cultured on an endothelial monolayer in the presence or absence of PP2. Cells were collected at 0 or 5 h, and immunoblots were probed against p-Src and Src. (F) Immunoblots showing the p-Src level in WT-Src and DN-Src transfectants. (G) Src transfectants were cultured on an endothelial monolayer, and cells were collected at 0 or 5 h for immunoblot analysis of p-Src. In F and G, arrowheads indicate WT- or DN-Src-GFP, whereas arrows indicate the endogenous Src.