Fig. 2 . Subcellular localization of CD8/GLG1 and CD8/GLG2 chimeric molecules. Human embryonic kidney epithelial 293 cells were stably transfected with pcDNA3.1 containing DNA sequences for the extracellular and transmembrane domain of CD8 (a), the extracellular domain of CD8 and transmembrane and intracellular domain of GLG1 (CD8/GLG1, b), or the extracellular domain of CD8 and transmembrane and intracellular domain of GLG2 (c-f). Transfected cells were fixed on coverslips, permeabilized with Triton X-100, and analyzed by immunofluorescence with FITC-conjugated monoclonal anti-human CD8. Giantin, a marker for Golgi apparatus, was detected with a rabbit polyclonal serum followed by Rhodamine-conjugated goat anti-rabbit immunoglobulin. In panel (d), CD8/GLG2 cells were treated with brefeldin A for 1 hour before fixation. In panel (e), Golgi structures were allowed to recover after brefeldin A treatment by incubation with media for 1 hour after washing out the brefeldin A. CD8/GLG2 cells in panel (f) were incubated with 5 µg/ml of cycloheximide for 4 hours before fixation. Images were collected with a Zeiss LSM510 laser-scanning confocal microscope. Data shown are the projection of scanned images of transfected cells.