Fig. 1. CD9 alters the localization and presentation of TGF. MDCK cells stably expressing CD9 or TGF, or coexpressing CD9 and TGF were analyzed. (A) Cells were fixed, permeabilized, stained and analyzed by confocal immunofluorescence microscopy (green, CD9; red, TGF). (B) Cell-surface immunostaining of TGF of MDCK cells that express TGF, or CD9 and TGF together. Top panels show cell-surface staining of non-permeabilized cells on ice, whereas the lower panels shows total immunofluorescence for TGF of permeabilized and fixed cells. The middle panels show phase-contrast microscopy of the stained cells in the top panels. (C) The cell-surface proteins labeled by biotinylation were subjected to immunoprecipitation for TGF, thus showing the level of cell-surface TGF in cells expressing CD9, TGF, or CD9 and TGF together (top panel). Middle and lower panels show immunoblotting for the expression levels of total TGF and CD9, respectively. Arrows in the middle panel mark the three differentially processed TGF forms (Bringman et al., 1987; Shi et al., 2000) (see also Fig. 2A). The arrow in the upper panel marks biotinylated cell-surface TGF, corresponding to unglycosylated transmembrane TGF without its pre-sequence (Bringman et al., 1987); this band corresponds to the lower one in the middle panel. (D) CD9 does not alter the distribution of TGF that lacks the TGF core sequence. MDCK cells stably expressing CD9, TGFE or both were fixed, permeabilized, stained and analyzed by confocal immunofluorescence microscopy (green, CD9; red, TGFE).