Fig. 5. Subcellular distribution and relocalization of endogenous Dishevelled in Saos-2 cells in response to Pax3 expression. Confocal images of methanol/acetone-fixed Ad-ß-gal-infected (A, middle panel) and Ad-Pax3^flag-infected (A, right panel and B) cells, at three days postinfection, and uninfected (A, left panel) cells. Methanol/acetone fixation results in complete extraction of virally encoded GFP (B, left panel), with minimal staining with control FITC- (B, middle panel) or Texas Red-conjugated (B, right panel) secondary antibodies. In control cells Dishevelled staining is punctate and cytoplasmic with prominent staining along stress fibers where Dishevelled colocalizes with that of F-actin (C, top row). In cells expressing Pax3, Dishevelled accumulates at the plasma membrane and at cell-cell junctions where it colocalizes with cortical actin bundles (A, right panel; and C, bottom row). In Pax3-expressing cells, Dishvelled also accumulates peripherally around the nucleus and at the tips of filopodia-like protrusions. Bars, 20 µm.