Figure 4. NEU3-HA-GFP specifically modifies the ganglioside pattern of non-DRM and DRM.

OFF HeLa tTA2 NEU3-HA-GFP cells were metabolically labeled for 2 h with [³H]Sphingosine and, after a 24 h chase, dox was removed for the indicated time periods. Cells were then extracted in the appropriate buffer containing 1% Triton X-100 for 30 min at 4°C. non-DRM (upper panel) and DRM (lower panel) were separated by Opti-Prep density gradient centrifugation. Equal aliquots of fractions 2 and 3 (DRM) and of fractions 6, 7 and 8 (non-DRM) were pooled and gangliosides and non-ganglioside sphingolipids were extracted, separated and quantified. Values are given as percentage of total and represent the means ± S.D. of 3 independent experiments. *p<0.05; **p<0.01; ***p<0.001.