Fig. 5. The integrity of syntaxin clusters depends on cholesterol. (A) Clusters disintegate upon cholesterol depletion.Unfixed membrane sheets were treated for 30 min at 37°C with 15 mM methyl-β-cyclodextrin and then immunostained for syntaxin 1. Upper row, membranes displaying increasing degrees of cyclodextrin-induced changes. Spots become less separated until a uniform distribution of the label is observed. Finally, membranes disintegrate. Boxes in the images indicate regions that are magnified below. (B and C) In vivo labeling of syntaxin 1 with photocholesterol. PC12 cells were photoaffinity labeled with either [³H]photocholesterol (B) or 10-azistearic acid/[³H]choline (C). Syntaxin was immunoprecipitated from detergent-extracted cell lysates using a monoclonal antibody against syntaxin 1. Aliquots of starting extracts (start), the supernatants after immunoprecipitation (unbound) and the immunoprecipitates (bound) were subjected to SDS–PAGE followed by blotting on nitrocellulose membrane. Left, autoradiographs (detected by phosphoimaging); right, immunoblots for syntaxin 1(using a polyclonal antibody) of the same membranes. For comparison, the starting material was adjusted to approximately equal amounts of radioactivity.