Fig. 1. Expression of FL-DG and ßDG in BAE. (A) Cells were transfected with pREP/F, pREP/C or vector alone and double-stained after geneticin selection with anti-ßDG and Texas-Red—phalloidin. Bound antibodies were visualized by a Cy2-conjugated secondary antibody. Serial optical sections (1 µm intervals) were obtained with a confocal microscope and through-focused images were reconstituted for the anti-ßDG images. At the laser intensity and the window level applied to reveal the distribution of expressed ßDG (FL-DG) and ßDG (ßDG), the signals from endogenous ßDG were extremely low. Subcellular distribution of endogenous ßDG (vector) in BAE has been described in detail (Shimizu et al., 1999). (B,C) Enrichment of FL-DG- or ßDG-expressing BAE. Cells were transfected and selected for resistance against geneticin and then processed for anti-ßDG flow cytometry (B) or western blotting (C). Note that each of the three types of transfected cells forms a single peak, indicating that it is a homogenious cell population (B). Arrowheads in (C) indicate intact ßDG (43 kDa) and ßDG (17 kDa). Molecular weights (kDa) are given on the left. Shown are the results obtained in a single experiment. Similar findings were obtained in two other independent experiments.