Active Rho is membrane-associated. (A) RhoA (Lulu51) and (B) monoclonal anti-RhoB (see Results) are apparent in the membrane of epithelial progenitors (arrows) where they co-localize with membrane-GFP, and progressively disappear from cells about to delaminate (arrowheads). Insets in (B) show cytoplasmic staining in a mesenchymal cell (M) but absence of membranous immunoreactivity when compared to GFP. All patterns are similar to that observed with the antibodies depicted in Figure 1. (C,D) In situ assay with the Rho-binding domain of Rothekin (RBD-GST). Note the membranal pattern apparent in epithelial cells co-localizing with membrane GFP (C, yellow in inset), and absence of cell surface pattern in explants treated with C3 transferase (D, and also inset showing green fluorescence of GFP but not of RBD-GST in the membranes). Bar = 7.5 μM.