Fig. 6. The V integrin subunit is expressed in epididymal epithelial cells and localizes to focal plaques in cells adherent to SED1. (A) Immunoblotting for V in the three epididymal regions; samples are loaded with equal protein. Immunoblotting of wild-type (+/+) and SED1-null (–/–) tissue under nonreducing conditions results in a single prominent band at 125 kDa, the reported molecular weight for V under nonreduced conditions. Wild-type and SED1-null tissue express V at similar levels. (B) Similarly, lysates of epithelial-enriched primary epididymal cells immunoprobed for V exhibit a single 125 kDa band. β-tubulin serves as a loading control. (C) Immunostaining of cells cultured on SED1 reveals cytokeratin-positive epididymal epithelial cells (red) that express V integrins (green) in punctate bundles arranged along the basal surface. (D) Desmin-positive (red) smooth muscle cells exhibit little or no V immunoreactivity. Nonimmune (NI) stained cells produce background immunoreactivity. All images are from parallel experiments imaged under identical conditions. (E) Confocal micrographs reveal that cytokeratin-positive (inserts) epithelial cells cultured on SED1 localize V to focal plaques along the lamellipodia (arrows). (F) Epithelial cells cultured on laminin also express V; however, the immunoreactivity is not distributed in focal plaques as on SED1 substrates but remains perinuclear. Scale bars: 20 µm.