FIG. 5.
Localization of fluorescent ceramide (C6-NBD-ceramide) in HeLa cells. (A) C6-NBD-ceramide (NBD-C6-Cer) at 10 μM rapidly accumulated on the plasma membrane and perinuclear region of HeLa cells 1 min after the treatment, and the fluorescence in the perinuclear region increased significantly until 10 min (top row). C6-ceramide (C6-Cer)-induced translocation of δPKC-GFP showed a similar time course to that of C6-NBD-ceramide (bottom row). (B) HeLa cells transfected with δPKC were fixed after treatment with 10 μM C6-NBD-ceramide for 20 min. Cells were immunostained with anti-δPKC monoclonal antibody and with Cy3-labeled IgG as secondary antibody to make the expressed δPKC visible. The localization of C6-NBD-ceramide (NBD) is shown in green (left) and of δPKC is shown in red (center). On the merged image, the overlapped signals of C6-NBD-ceramide and Cy3 appear in yellow (right). The results shown are representative of three independent experiments. Bars, 10 μm (A, top row) and 20 μm (A, bottom row, and B).