Figure 5

Immunofluorence- and immunoprecipitation-based analyses of cellular localization of wild-type and mutant forms of the maxi-K channel in mouse fibroblasts. Cellular localization of (A) WT, (B) mutant F1012A, (C) mutant Y1007A, F1012A and (D) mutant Y1007A, F1012A, Y1015A maxi-K channel forms in Ltk- cells, as detected by immunocytochemistry, using antibodies against the maxi-K channel (red) and cav-1 (green). Images were pseudocolored for better comparison. WT and all mutant channels except mutant Y1007A, F1012A, Y1015A co-localized with cav-1 on the membrane (yellow). Data shown are representative examples of 6 (WT, mutant F1012A, mutant Y1007A, F1012A) or 5 (mutant Y1007A, F1012A, Y1015A) independent experiments. (E) Immunoprecipitation of the lipid raft fractions shown in the boxed region of Figure 4 (using an anti Maxi-K channel antibody) demonstrated that WT, mutant F1012A, and mutant Y1007A, F1012A, but not mutant Y1007A, F1012A, Y1015A, associate with cav-1 (E). Data shown are representative of 8 (WT), 10 (mutant F1012A), 9 (mutant Y1007A, F1012A) and 6 (mutant Y1007A, F1012A, Y1015A) independent experiments.