Fig. 2. Syndecan-4 is internalized from lipid raft regions of the plasma membrane. Confocal microscopy of syndecan-4 endocytosis was carried out in RFPEC expressing the FcR-S4 chimera. The raft portion of the plasma membrane was labeled with BODIPY GM1 (blue) (a) or by transient expression of GPI-GFP construct (f). FcR-S4 chimeras were decorated with biotinylated niIgG and clustering was then initiated by the addition of Cy3-F(ab')2 fragments (red). Five minutes after the initiation of antibody clustering, all internalized FcR-S4 chimeras (b) were seen in close association with GM1-BODIPY (magenta, a,d,c) membrane domains or with GPI-GFP (yellow, h and i). (e,i) Merged color images (d and h, respectively) overlaid with DIC images. The depletion of membrane cholesterol with MßCD fully blocks clustering-induced FcR-S4 internalization (j,k,l,m). Scale bars, 10 µm.