Abstract
This chapter presents a methodological approach to the study of adenosine diphosphate (ADP)-ribosylation factor (ARF) and its regulation of spectrin follows three experimental strategies: (1) the use of expressed spectrin peptides in cultured cell lines, observed by indirect immunofluorescent microscopy, to identify the spectrin sequence motifs responsible for targeting it to the Golgi or other organelles in vivo. (2) An immunofluorescence analysis of changes in the intracellular distribution of spectrin in permeabilized cultured cells in response to activators (GTPTS) or inhibitors (brefeldin A, BFA) of ARF. (3) In vitro assays of the binding of spectrin or recombinant spectrin peptides to isolated Golgi membranes. Typically, these approaches utilize recombinant βI- or βIII-spectrin peptides representing different functional domains of spectrin [epitope tagged or green fluorescent protein (GFP) labeled], or antibodies specific for βI- or βIII spectrin. Also required are methods for introducing these peptides into cells, methods for preparing isolated Golgi membranes, and methods for manipulating the activity or abundance of ARF.
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