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Fig. 7. 2-Bromopalmitate suppresses the mislocalization phenotype of selected mutants. (A) PC12 cells were transfected with full-length yeast Ykt6p mutants. At 5 hours after transfection, the growth medium was changed to a medium containing 2-bromopalmitate and incubated until the cells were fixed. After fixation, subcellular localization of each mutant and endogenous rat Ykt6 was examined by double-labeling fluorescent microscopy using anti-Myc antibody and anti-Ykt6 antibody. Top panels show co-staining of endogenous Ykt6 (left) and Myc-Ykt6p-V8N (middle), and merged (right) images after drug treatment. Arrows point to puncta where colocalization is observed (27% overlap). The same degree of colocalization was observed for Myc-Ykt6p-K100E/Y101N (bottom left) and endogenous Ykt6 (not shown). Mislocalization phenotypes of Myc-Ykt6p-F42E (bottom center), Myc-Ykt6p F39E (bottom right), Myc-Ykt6p R50E/R56E (not shown) and Myc-Ykt6p I59E (not shown) were not altered by the 2-bromopalmitate treatment. Scale bar, 10 µm. (B) GAP-43 translocates from membrane to cytosol in the presence of 2-bromopalmitate. Localization of endogenous GAP-43 was analysed by membrane fractionation followed by immunoblotting. Abbreviations: P, pellet fraction; S, soluble fraction; T, total fraction. Presence or absence of 2-bromopalmitate (2-BP) is indicated at the top.
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