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Fig. 3. Effect of ceramide on HERG protein expression. (A) HERG protein expression was analysed by western blot. Ceramide-treated (10 µM for 60 minutes) cells were lysed and ultracentrifuged as described in Materials and Methods and proteins were separated by 6% SDS-PAGE. HERG protein was detected with monoclonal rabbit anti-HERG (1:1000). The higher molecular weight band (155 kDa) corresponds to the fully glycosylated mature HERG protein and the lower molecular weight band (135 kDa) corresponds to the core-glycosylated immature HERG protein. Results of the densitometric analysis are shown in the lower panel. The results represent the mean±s.e.m. of nine separate experiments. (B) Specific cell surface expression of HERG protein was analysed by biotin labelling. After incubation with ceramide cell surface proteins were biotinylated with a biotinylating reagent, sulfo-NHS-SS-biotin. HERG protein was immunoprecipitated and the biotinylated HERG channels were detected by horseradish peroxidase conjugated streptavidin. The blot shown is a representative of three separate experiments.
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