Figure 2. Evidence that a Xentry-β-galactosidase conjugate is internalized by living HepG2 cells, and that Xentry enters cells via a syndecan pathway.

(a) Visualization of the internalization of an unlabelled Xentry-β-galactosidase conjugate, and unconjugated β-galactosidase, after a 10 min incubation with live cells preloaded with the green fluorescent substrate C₁₂FDG. (b) Confocal microscopy reveals the Xentry-β-galactosidase conjugate is taken up into the cytoplasm, but excluded from the nucleus. (c) Uptake of FITC-labelled Xentry (10 μM) by HepG2 cells is blocked by treatment of cells with 1 μg/ml of anti-syndecan-4 antibody, whereas 1 μg/ml of a control antibody against TRAIL has no effect. (d) Syndecan-deficient K562 cells transfected to express syndecan-4 show increased uptake of TAMRA-labelled (red) Xentry (20 μM). Syndecan-4-expressing transfectants were detected with a rabbit polyclonal anti-syndecan 4 antibody, followed by a FITC-labelled (green) donkey anti-rabbit antibody. Cell nuclei were stained blue with DAPI. Arrows mark syndecan 4-expressing transfectants that have taken up TAMRA-labelled Xentry. Scale bar, 50 μm.