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Fig. 4. Effect of ceramide on HERG cell surface expression. (A) Ceramide-induced internalisation of HERG channels in HEK293 cells. In quantitative analysis of anti-HERG immunoreactivity statistically significant changes in plasma membrane and cytosolic immunofluorescence levels were observed after 30 minutes of ceramide treatment. Results are shown relative to nuclear fluorescence levels at baseline. Each bar gives the mean±s.e.m. of seven separate experiments. (B) Confocal sections of HEK cells labelled with anti-HERG antibody. Ceramide treatment for 60 minutes induced a clear internalisation of immunoreactivity from plasma membrane to cytosol. This internalisation was inhibited by the lysosomal inhibitor bafilomycin and by low-temperature treatment (16°C, 45 minutes), which curtails endocytosis. In contrast, the ceramide-induced internalisation was not inhibited by the proteosomal inhibitor MG132. The figures shown are representative cells of three to ten separate experiments. Bar, 10 µM.





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