Fig. 5. Ubiquitylation of the HERG channel in HEK293 cells. (A) HERG-expressing HEK293 cells were exposed to ceramide 10 µM (60 minutes) and the HERG protein was immunoprecipitated. The amount of ubiquitin in HERG protein was determined by western blotting using an anti-ubiquitin antibody (clone P4G7) recognising both poly- and monoubiquitylated proteins. The same blot was also stained for HERG showing the levels of HERG protein in the cell lysates. The intensity of the bands was analysed and the bars show the ratio of ubiquitin and HERG in control conditions and in cells stimulated with 10 µM ceramide for 1 hour. The normalised values represent the mean±s.e.m. of five experiments. (B) Prior to treatment with 10 µM ceramide (60 minutes), HERG-expressing HEK293 cells were exposed for 60 minutes to either 20 µM MG132 or 0.25 µM bafilomycin. HERG protein was immunoprecipitated and the amount of ubiquitin was determined by western blot using an anti-ubiquitin antibody (clone P4G7). The bar graphs show the intensity of the bands as a ratio of ubiquitin and HERG. The normalised values represent the mean±s.e.m. of three experiments. (C) Downregulation of HERG through the lysosomal pathway. Control cells and ceramide-treated cells (10 µM for 60 minutes) were co-stained with antibodies for the lysosomal marker Lamp-1 (red) and HERG protein (green). The colocalisation is seen as yellow. The figures shown are representative cells of three separate experiments. Bar, 10 µM.