Localization of CD317 in polarized Caco-2 cells, its knockdown in Caco-2 cells, and the effect of knockdown on F-actin. (A) Localization of CD317 in polarized Caco-2 cells. The top row shows XY images of CD317 (detected with an anti-CD317 antibody) and F-actin (Alexa Fluor 594–phalloidin) localization as indicated. The second row shows an XZ section from the first row. The third row shows XY images of CD317 and β-catenin localization as indicated (β-catenin is a marker of lateral membranes). The bottom row shows an XZ section from the third row. Panels on the right show merged images of the left and middle panels, with DAPI-stained nuclei on the right in the second row. (B) Immunoblot analysis (using an anti-CD317 antibody) of lysates from Caco-2 cells stably expressing CD317 siRNA or control GFP siRNA as indicated. The bands representing higher molecular weight proteins in the control siRNA lane correspond to glycosylated CD317. An immunoblot of α-tubulin was used as a loading control. Molecular mass is indicated in kilodaltons. (C) Alexa Fluor 594–phalloidin decoration of F-actin in nonpolarized CD317 knockdown cells (top) and control cells (bottom). Each image shows a colony of cells with DAPI-stained nuclei. The top panel is an XZ section from a field of CD317 knockdown cells. Bars, 10 µm.